Immunoregulatory peptides and methods of use

ABSTRACT

Peptides for the treatment of inflammation, and therapeutic uses and methods of using the same are disclosed. Peptides including a transducing sequence are effective for inhibiting cytokine activity and TNF-α secretion through interaction with toll-like receptors. Experiments are described illustrating the efficacy of the compounds in treating otitis media.

This application is a divisional of U.S. patent application Ser. No.12/794,185, filed Jun. 4, 2010, which claims the priority benefit ofU.S. Provisional Application No. 61/184,438, filed Jun. 5, 2009, U.S.Provisional Application No. 61/184,455, filed Jun. 5, 2009, U.S.Provisional Application No. 61/220,738, filed Jun. 26, 2009, U.S.Provisional Application No. 61/220,745, filed Jun. 26, 2009, and U.S.Provisional Application No. 61/256,364, filed Oct. 30, 2009, each ofwhich is incorporated herein by reference in its entirety. Thisapplication contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jan. 23, 2013, is named37501703401_sequence_listing.txt and is 116 kilobytes in size.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The work leading to the present invention was supported by SBIRAI065000, “Bacterial-Induced Sepsis: A New Treatment Strategy” and SBIRDC005882, “New Treatment for Inflammation in Middle Ear Infections.” TheU.S. Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) recognize and respond to conserved motifstermed pathogen-associated molecular patterns (PAMPs). TLRs arecharacterized by an extracellular leucine-rich repeat motif and anintracellular Toll/IL-1 receptor (TIR) domain. Triggering of TLRs byPAMPs initiates a series of intracellular signaling events resulting inan inflammatory immune response designed to contain and eliminate thepathogen. Viruses encode immunoregulatory proteins, such as A52R(produced by the vaccina virus), that can effectively inhibitintracellular TIR signaling resulting in a diminished inflammatoryimmune response.

Chronic otitis media (COM) affects both children and adults. The chronicinflammation seen in COM can impact the inner ear and can lead tosensorineural hearing loss. COM is a significant medical problem, and nocurrent therapeutics, other than steroids, are available for treatingCOM. Many patients with COM require surgical placement of ear tubes. Aswith acute otitis media (AOM), patients with COM experience decreasedhearing due to development of an intense inflammatory response withinthe middle ear, including the presence of residual fluid.

SUMMARY OF THE INVENTION

In some embodiments, the invention contemplates a pharmaceuticalcomposition comprising a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue.

In some embodiments, the invention contemplates a pharmaceuticalcomposition comprising a peptide comprising a sequence of any one of SEQID NOS: 1-186.

In some embodiments, the invention contemplates a peptide comprising thesequence of any one of SEQ ID NOS: 1-368.

In some embodiments, the invention contemplates a derivative of apeptide comprising the sequence of any one of SEQ ID NOS: 1-369, whereinthe derivative comprises at least one D-amino acid residue.

In some embodiments, the invention contemplates a method of regulatingcellular activity, the method comprising administering to an organism inneed or want thereof an effective amount of a pharmaceutical compositioncomprising a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a method of treatinginflammation in an animal, the method comprising administering to ananimal in need or want thereof a pharmaceutical composition comprising apeptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a method of treatingsinusitis, the method comprising administering an aerosol composition toan organism in need or want thereof, the aerosol composition comprisinga therapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a method of improvinghearing in an animal, the method comprising administering to an animalhaving middle and/or inner ear inflammation and reduced hearing atherapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus, wherein the peptide is administered topically,wherein the hearing improves to a level no better than ordinary levels,and/or the hearing improves faster than the hearing would improvewithout administration of the peptide.

In some embodiments, the invention contemplates a method of treatingmiddle and/or inner ear inflammation, the method comprisingadministering to a tympanic membrane of an animal in need or wantthereof a therapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for regulating cellular activity, thepeptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating inflammation in an animal,the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating sinusitis, the medicamentcomprising an aerosol composition, the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for improving hearing in an animal,wherein the animal has middle and/or inner ear inflammation and reducedhearing, the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus, wherein the medicament is a topical medicament.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating middle and/or inner earinflammation, wherein the medicament is suitable for administration to atympanic membrane of an animal in need or want thereof, the peptidecomprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

DESCRIPTION OF THE FIGURES

FIG. 1: Illustrates, in Panels A and B, an assay for crossing thetympanic membrane by FITC-labeled P13. Panel A. Bright field microscopydemonstrates cells in middle ear fluid. Panel B. Fluorescent microscopydemonstrates FITC-labeled P13 associated with cells in middle ear fluid.

FIG. 2: P13 reduces middle ear inflammation. BALB/c mice (n=6 in PBSgroup and n=7 in P13 group) were injected with heat-killed S. pneumonia,treated 24 hours later with topical (ear drop) administration of P13 (1μg), and histology was examined 72 hours after bacteria introduction.Panel A: cell number within middle ear; Panel B: fluid area withinmiddle ear. Note: Animal with high cell number and high fluid area inthe P13 treated group is the same animal.

FIG. 3: Topical administration of P13 significantly improves hearingthresholds. BALB/c mice were administered heat-killed S. pneumonia, 24hours later mice were treated topically (ear drops) with P13 (1 μg) orPBS. Panel A: Hearing thresholds at 4, 8, 16 and 32 kHz were quantifiedby ABR at days 5 and 13 days after administration of bacteria. Hearingloss was calculated by subtracting the background ABR frompost-treatment ABR and summing across frequencies. Panel B: Number ofanimals with >20 DB hearing loss across all frequencies. Number ofanimals is in parentheses.

FIG. 4: Percent Inhibition of TNF-α secretion by peptides T52, S5 andP13. RAW264.7 cells were plated at 3×10⁵ cells/well in 48-well plates.After 24 h the cells were incubated with peptide at variousconcentrations at room temperature in triplicate for 15 minutes and thenstimulated with 1 μg/ml CpG-ODN. Cells were then incubated for 4 hoursat 37° C., supernatants collected, and TNF-α measured by ELISA. Percentinhibition was calculated by comparing TNF-α secretion from cellsincubated with peptide to control cells with no peptide treatment.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The treatment and control of natural and pathogen-induced inflammationrepresents a significant clinical challenge. The targeting of theTLR/TIR signaling cascade represents one approach to controlinflammation; thus the identification of peptides derived from the A52Rprotein or A52R-like proteins finds therapeutic applications. Thepeptides and pharmaceutical compositions of the invention disclosedherein, and uses and methods of using the same, present a solution tothe problem of controlling inflammation and regulating cellular pathwaysassociated with inflammation.

Throughout the disclosure, amino acid residues of the peptides of theinvention are referenced by one or both of the standard abbreviationsknown in the art: a) single-letter abbreviations, such as R forarginine, D for aspartic acid, V for valine, etc.; and b) three-letterabbreviations, such as Arg for arginine, Asp for aspartic acid, Val forvaline, etc. The invention contemplates both L- and D-forms of aminoacid residues. In cases wherein the abbreviation refers only to an aminoacid residue of the D-configuration, the abbreviation is preceded by theterm, “D-,” for example, D-Arg for D-arginine, D-Asp for D-asparticacid, D-Val for D-valine, etc.

Toll-Like Receptor Signaling

Toll-Like receptors (“TLRs”) are conserved molecular receptors thatrecognize structures from bacteria, fungi, protozoa, and viruses.Activation of TLRs initiates a series of intracellular events resultingin an innate immune response characterized by the production ofpro-inflammatory cytokines (References 2-9). TLR signaling originatesfrom the cytoplasmic Toll/interleukin-1 receptor (TIR) domain, conservedamong all TLRs. Not limited by any theory, in certain embodiments,adapter molecule MyD88, containing both a TIR domain and a death domain,can associate with the TIR domain of TLRs and IRAK proteins.Phosporylation of IRAK can then lead to association with TRAF6 andsubsequent activation of NF-κB and secretion of pro-inflammatorycytokines (References 14, 22-25).

Peptides

Vaccinia virus, a member of the poxvirus family, is a DNA virus that hasbeen demonstrated to encode immunomodulatory proteins (References15-18). One of these proteins, A52R, has been shown to inhibit NF-κBactivation following initiation of the TIR signaling cascade (References15 and 18). Recent studies have demonstrated that A52R inhibits TRsignaling and contributes to the virulence of vaccinia virus. In certainembodiments, cell activation in response to different PAMPs involves anumber of intracellular molecules common to all TLRs, including but notlimited to MyD88, members of the IL-1 receptor-associated kinase (IRAK)proteins, TNF receptor associated factor (TRAF6), and NF-κB (Reference1).

Harte and colleagues (Reference 18) have demonstrated that the A52Rprotein inhibits TIR signaling by binding to both IRAK2 and TRAF6.Deletion of the A52R protein from vaccinia virus results in reducedviral virulence.

The peptide 13 (“P13”) sequence (DIVKLTVYDCI (SEQ ID NO: 369)) wasderived from the A52R sequence from vaccinia virus. Blast searchanalysis shows that peptide P13 has 100% homology with sequences foundwithin larger proteins from vaccinia virus other than A52R, two proteinsfrom cowpox virus, and one protein from rabbit pox virus. Peptide 13 wasshown to have significant homology with three separate proteins fromdifferent strains of variola (smallpox) virus: i) A46L from variolamajor virus strain India; ii) A49L from variola minor virus Garcia; andiii) A44L from variola major virus strain.

P13 inhibits toll-like receptor-dependent signaling (U.S. Pat. No.7,192,930 and US2008/0039395 incorporated herein by reference in theirentirety). In some embodiments, structure-activity testing can beperformed to identify amino acid residues in the P13 sequence that canbe substituted for enhanced activity.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a peptide derived from A52R. In some embodiments,the pharmaceutical composition is an aural pharmaceutical composition.

In some embodiments, the peptide is P13. In some embodiments, thepeptide is derived from P13. In some embodiments, the peptide is a P13variant, derivative, stereoisomer, or analogue. In some embodiments, thepeptide comprises the amino acid sequence LEEYFMY (SEQ ID NO: 370). Insome embodiments, the peptide comprises the amino acid sequenceFTILEEYFMY (SEQ ID NO: 371). In some embodiments, the peptide comprisesthe amino acid sequence DIVKLTVYDCI (SEQ ID NO: 369). In someembodiments, the peptide comprises the amino acid sequence VYDCI (SEQ IDNO: 372). In some embodiments, the peptide comprises the amino acidsequence VYACI (SEQ ID NO: 373). In some embodiments, the peptidecomprises the amino acid sequence KLTVY (SEQ ID NO: 374). In someembodiments, the peptide comprises the amino acid sequence KLYVY (SEQ IDNO: 375). In some embodiments, the peptide comprises the amino acidsequence KVYVY (SEQ ID NO: 376). In some embodiments, the peptidecomprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. In some embodiments,the peptide comprises from 7-50 residues.

In some embodiments, a peptide comprises a transducing sequence. Thetransducing sequence can participate in cellular uptake. The presence ofthe transducing sequence can lead to enhanced or selective cellularuptake. In some embodiments, the transducing sequence is at theN-terminus of the peptide. In some embodiments, the transducing sequenceis at the C-terminus of the peptide.

A non-limiting example of a transducing sequence is a poly-argininesequence. In some embodiments, the poly-arginine sequence comprisesarginine residues. In some embodiments, the poly-arginine sequenceconsists of arginine residues. In specific embodiments, the transducingsequence comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20arginine residues. In some embodiments, the transducing sequencecomprises nine arginine residues (SEQ ID NO: 379). In some embodiments,the transducing sequence consists of nine arginine residues (SEQ ID NO:379). The arginine residues of the transducing sequence can beL-arginine, D-arginine, or a mixture of L- and D-arginine.

Table 1 provides non-limiting examples of peptides of the invention.Exemplary peptides 1-130 are those derived from A52R. Of peptides 1-130,a subset is further designated S1-S22. Other examples include peptidesderived from P13 (“T peptides”). In some embodiments, the presentinvention provides a pharmaceutical composition comprising a peptidecomprising any amino acid sequence listed in Table 1. In someembodiments, the present invention provides a pharmaceutical compositioncomprising any peptide listed in Table 1. In some embodiments, thepresent invention provides a pharmaceutical composition comprising apeptide comprising any amino acid sequence of S1-S22. In someembodiments, the present invention provides a pharmaceutical compositioncomprising any peptide of S1-S22. In some embodiments, the presentinvention provides a pharmaceutical composition comprising a peptidecomprising any amino acid sequence of T1-T56. In some embodiments, thepresent invention provides a pharmaceutical composition comprising anypeptide of T1-T56. In some embodiments, the peptide has a transducingsequence. In some embodiments, the transducing sequence ispoly-arginine. In some embodiments, the peptide comprises the sequenceof any one of SEQ ID NOS: 42-44, 68-77, 79-81, 83, 102-106, 133, 141,151, 166, 167, 181, 182, 228-230, 254-263, 265-267, 269, 288-292, 319,327, 337, 352, 353, 367, and 368. In some embodiments, the peptidecomprises the sequence of T3, T11, T21, T36, T37, T51, or T52. In someembodiments, the peptide comprises the sequence of T3-R⁹, T11-R⁹,T21-R⁹, T36-R⁹, T37-R⁹, T51-R⁹, or T52-R⁹.

TABLE 1  Exemplary peptides of the invention Peptide Sequence   1-R⁹YIKVQKQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 1)   2-R⁹IKVQKQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 2)   3-R⁹KVQKQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 3)   4-R⁹VQKQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 4)   5-R⁹QKQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 5)   6-R⁹KQDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 6)   7-R⁹QDIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 7)   8-R⁹DIVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 8)   9-R⁹IVKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 9)  10-R⁹VKLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 10)  11-R⁹KLTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 11)  12-R⁹LTVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 12)  13-R⁹TVYDCISMIGLCARRRRRRRRR (SEQ ID NO: 13)  14-R⁹VYDCISMIGLCARRRRRRRRR (SEQ ID NO: 14)  15-R⁹YDCISMIGLCARRRRRRRRR (SEQ ID NO: 15)  16-R⁹DCISMIGLCARRRRRRRRR (SEQ ID NO: 16)  17-R⁹CISMIGLCARRRRRRRRR (SEQ ID NO: 17)  18-R⁹ISMIGLCARRRRRRRRR (SEQ ID NO: 18)  19-R⁹SMIGLCARRRRRRRRR (SEQ ID NO: 19)  20-R⁹ MIGLCARRRRRRRRR (SEQ ID NO: 20) 21-R⁹ IGLCARRRRRRRRR (SEQ ID NO: 21)  22-R⁹YIKVQKQDIVKLTVYDCISMIGLCRRRRRRRRR (SEQ ID NO: 22)  23-R⁹YIKVQKQDIVKLTVYDCISMIGLRRRRRRRRR (SEQ ID NO: 23)  24-R⁹YIKVQKQDIVKLTVYDCISMIGRRRRRRRRR (SEQ ID NO: 24)  25-R⁹YIKVQKQDIVKLTVYDCISMIRRRRRRRRR (SEQ ID NO: 25)  26-R⁹YIKVQKQDIVKLTVYDCISMRRRRRRRRR (SEQ ID NO: 26)  27-R⁹YIKVQKQDIVKLTVYDCISRRRRRRRRR (SEQ ID NO: 27)  28-R⁹YIKVQKQDIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 28)  29-R⁹YIKVQKQDIVKLTVYDCRRRRRRRRR (SEQ ID NO: 29)  30-R⁹YIKVQKQDIVKLTVYDRRRRRRRRR (SEQ ID NO: 30)  31-R⁹YIKVQKQDIVKLTVYRRRRRRRRR (SEQ ID NO: 31)  32-R⁹YIKVQKQDIVKLTVRRRRRRRRR (SEQ ID NO: 32)  33-R⁹YIKVQKQDIVKLTRRRRRRRRR (SEQ ID NO: 33)  34-R⁹YIKVQKQDIVKLRRRRRRRRR (SEQ ID NO: 34)  35-R⁹YIKVQKQDIVKRRRRRRRRR (SEQ ID NO: 35)  36-R⁹YIKVQKQDIVRRRRRRRRR (SEQ ID NO: 36)  37-R⁹YIKVQKQDIRRRRRRRRR (SEQ ID NO: 37)  38-R⁹YIKVQKQDRRRRRRRRR (SEQ ID NO: 38)  39-R⁹YIKVQKQRRRRRRRRR (SEQ ID NO: 39)  40-R⁹ YIKVQKRRRRRRRRR (SEQ ID NO: 40) 41-R⁹ YIKVQRRRRRRRRR (SEQ ID NO: 41)  42 (S1)-R⁹EMFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 42)  43 (S2)-R⁹MFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 43)  44 (S3)-R⁹FTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 44)  45-R⁹TILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 45)  46-R⁹ILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 46)  47-R⁹LEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 47)  48-R⁹EEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 48)  49-R⁹EYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 49)  50-R⁹YFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 50)  51-R⁹FMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 51)  52-R⁹MYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 52)  53-R⁹YRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 53)  54-R⁹RGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 54)  55-R⁹GLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 55)  56-R⁹LLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 56)  57-R⁹LGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 57)  58-R⁹GLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 58)  59-R⁹LRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 59)  60-R⁹RIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 60)  61-R⁹IKYGRLFNEIRRRRRRRRR (SEQ ID NO: 61)  62-R⁹KYGRLFNEIRRRRRRRRR (SEQ ID NO: 62)  63-R⁹YGRLFNEIRRRRRRRRR (SEQ ID NO: 63)  64-R⁹GRLFNEIRRRRRRRRR (SEQ ID NO: 64)  65-R⁹ RLFNEIRRRRRRRRR (SEQ ID NO: 65) 66-R⁹ LFNEIRRRRRRRRR (SEQ ID NO: 66)  67-R⁹EMFTILEEYFMYRGLLGLRIKYGRLFNERRRRRRRRR (SEQ ID NO: 67)  68 (S4)-R⁹EMFTILEEYFMYRGLLGLRIKYGRLFNRRRRRRRRR (SEQ ID NO: 68)  69 (S5)-R⁹EMFTILEEYFMYRGLLGLRIKYGRLFRRRRRRRRR (SEQ ID NO: 69)  70 (S6)-R⁹EMFTILEEYFMYRGLLGLRIKYGRLRRRRRRRRR (SEQ ID NO: 70)  71 (S7)-R⁹EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRRR (SEQ ID NO: 71)  72 (S8)-R⁹EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 72)  73 (S9)-R⁹EMFTILEEYFMYRGLLGLRIKYRRRRRRRRR (SEQ ID NO: 73)  74 (S10)-R⁹EMFTILEEYFMYRGLLGLRIKRRRRRRRRR (SEQ ID NO: 74)  75 (S11)-R⁹EMFTILEEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 75)  76 (S12)-R⁹EMFTILEEYFMYRGLLGLRRRRRRRRRR (SEQ ID NO: 76)  77 (S13)-R⁹EMFTILEEYFMYRGLLGLRRRRRRRRR (SEQ ID NO: 77)  78-R⁹EMFTILEEYFMYRGLLGRRRRRRRRR (SEQ ID NO: 78)  79 (S14)-R⁹EMFTILEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 79)  80 (S15)-R⁹EMFTILEEYFMYRGLRRRRRRRRR (SEQ ID NO: 80)  81 (S16)-R⁹EMFTILEEYFMYRGRRRRRRRRR (SEQ ID NO: 81)  82-R⁹EMFTILEEYFMYRRRRRRRRRR (SEQ ID NO: 82)  83 (S17)-R⁹EMFTILEEYFMYRRRRRRRRR (SEQ ID NO: 83)  84-R⁹EMFTILEEYFMRRRRRRRRR (SEQ ID NO: 84)  85-R⁹EMFTILEEYFRRRRRRRRR (SEQ ID NO: 85)  86-R⁹EMFTILEEYRRRRRRRRR (SEQ ID NO: 86)  87-R⁹EMFTILEERRRRRRRRR (SEQ ID NO: 87)  88-R⁹EMFTILERRRRRRRRR (SEQ ID NO: 88)  89-R⁹ EMFTILRRRRRRRRR (SEQ ID NO: 89) 90-R⁹ EMFTIRRRRRRRRR (SEQ ID NO: 90)  91-R⁹MFTILLEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 91)  92-R⁹FTILLEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 92)  93-R⁹TILLEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 93)  94-R⁹ILLEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 94)  95-R⁹LLEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 95)  96-R⁹LEEYFMYRGLLRRRRRRRRR (SEQ ID NO: 96)  97-R⁹EYFMYRGLLRRRRRRRRR (SEQ ID NO: 97)  98-R⁹YFMYRGLLRRRRRRRRR (SEQ ID NO: 98)  99-R⁹FMYRGLLRRRRRRRRR (SEQ ID NO: 99) 100-R⁹ MYRGLLRRRRRRRRR (SEQ ID NO: 100)101-R⁹ YRGLLRRRRRRRRR(SEQ ID NO: 101) 102 (S18)-R⁹MFTILEEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 102) 103 (S19)-R⁹FTILLEEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 103) 104 (S20)-R⁹TILLEEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 104) 105 (S21)-R⁹ILLEEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 105) 106 (S22)-R⁹LLEEYFMYGLLGLRIRRRRRRRRR (SEQ ID NO: 106) 107-R⁹EYFMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 107) 108-R⁹YFMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 108) 109-R⁹FMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 109) 110-R⁹MYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 110) 111-R⁹YRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 111) 112-R⁹RGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 112) 113-R⁹GLLGLRIKYGRRRRRRRRR (SEQ ID NO: 113) 114-R⁹LLGLRIKYGRRRRRRRRR (SEQ ID NO: 114) 115-R⁹LGLRIKYGRRRRRRRRR (SEQ ID NO: 115) 116-R⁹GLRIKYGRRRRRRRRR (SEQ ID NO: 116) 117-R⁹LRIKYGRRRRRRRRR (SEQ ID NO: 117) 118-R⁹ RIKYGRRRRRRRRR (SEQ ID NO: 118)119-R⁹ EEYFMRRRRRRRRR (SEQ ID NO: 119) 120-R⁹EEYFMYRRRRRRRRR (SEQ ID NO: 120) 121-R⁹EEYFMYRRRRRRRRRR (SEQ ID NO: 121) 122-R⁹EEYFMYRGRRRRRRRRR (SEQ ID NO: 122) 123-R⁹EEYFMYRGLRRRRRRRRR (SEQ ID NO: 123) 124-R⁹EEYFMYRGLLRRRRRRRRR (SEQ ID NO: 124) 125-R⁹EEYFMYRGLLGRRRRRRRRR (SEQ ID NO: 125) 126-R⁹EEYFMYRGLLGLRRRRRRRRR (SEQ ID NO: 126) 127-R⁹EEYFMYRGLLGLRRRRRRRRRR (SEQ ID NO: 127) 128-R⁹EEYFMYRGLLGLRIRRRRRRRRR (SEQ ID NO: 128) 129-R⁹EEYFMYRGLLGLRIKRRRRRRRRR (SEQ ID NO: 129) 130-R⁹EEYFMYRGLLGLRIKYRRRRRRRRR (SEQ ID NO: 130)  T1-R⁹IVKLTVYDCIRRRRRRRRR (SEQ ID NO: 131)  T2-R⁹DIVKLTVYDCRRRRRRRRR (SEQ ID NO: 132)  T3-R⁹AIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 133)  T4-R⁹DAVKLTVYDCIRRRRRRRRR (SEQ ID NO: 134)  T5-R⁹DIAKLTVYDCIRRRRRRRRR (SEQ ID NO: 135)  T6-R⁹DIVALTVYDCIRRRRRRRRR (SEQ ID NO: 136)  T7-R⁹DIVKATVYDCIRRRRRRRRR (SEQ ID NO: 137)  T8-R⁹DIVKLAVYDCIRRRRRRRRR (SEQ ID NO: 138)  T9-R⁹DIVKLTAYDCIRRRRRRRRR (SEQ ID NO: 139) T10-R⁹DIVKLTVADCIRRRRRRRRR (SEQ ID NO: 140) T11-R⁹DIVKLTVYACIRRRRRRRRR (SEQ ID NO: 141) T12-R⁹DIVKLTVYDAIRRRRRRRRR (SEQ ID NO: 142) T13-R⁹DIVKLTVYDCARRRRRRRRR (SEQ ID NO: 143) T14-R⁹EIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 144) T15-R⁹DIVKLTVYECIRRRRRRRRR (SEQ ID NO: 145) T16-R⁹DIVRLTVYDCIRRRRRRRRR (SEQ ID NO: 146) T17-R⁹DIVHLTVYDCIRRRRRRRRR (SEQ ID NO: 147) T18-R⁹KIVKLTVYKCIRRRRRRRRR (SEQ ID NO: 148) T19-R⁹DIVELTVYDCIRRRRRRRRR (SEQ ID NO: 149) T20-R⁹DIVKLSVYDCIRRRRRRRRR (SEQ ID NO: 150) T21-R⁹DIVKLYVYDCIRRRRRRRRR (SEQ ID NO: 151) T22-R⁹DIVKLTVSDCIRRRRRRRRR (SEQ ID NO: 152) T23-R⁹DIVKLTVTDCIRRRRRRRRR (SEQ ID NO: 153) T24-R⁹DIVKLTVWDCIRRRRRRRRR (SEQ ID NO: 154) T25-R⁹DIVKLTVFDCIRRRRRRRRR (SEQ ID NO: 155) T26-R⁹DIVKLTVYDMIRRRRRRRRR (SEQ ID NO: 156) T27-R⁹DIVKLTVYDSIRRRRRRRRR (SEQ ID NO: 157) T28-R⁹DIVKLTVYDXaaIRRRRRRRRR (SEQ ID NO: 158) T29-R⁹DIVKLTVYDXaaIRRRRRRRRR (SEQ ID NO: 159) T30-R⁹DIVKLTVYDXaaIRRRRRRRRR (SEQ ID NO: 160) T31-R⁹DIVKLTVYDXaaIRRRRRRRRR (SEQ ID NO: 161) T32-R⁹DIVKLTVYDXaaIRRRRRRRRR (SEQ ID NO: 162) T33-R⁹DLVKLTVYDCIRRRRRRRRR (SEQ ID NO: 163) T34-R⁹DVVKLTVYDCIRRRRRRRRR (SEQ ID NO: 164) T35-R⁹DILKLTVYDCIRRRRRRRRR (SEQ ID NO: 165) T36-R⁹DIIKLTVYDCIRRRRRRRRR (SEQ ID NO: 166) T37-R⁹DIVKVTVYDCIRRRRRRRRR (SEQ ID NO: 167) T38-R⁹DIVKITVYDCIRRRRRRRRR (SEQ ID NO: 168) T39-R⁹DIVKLTLYDCIRRRRRRRRR (SEQ ID NO: 169) T40-R⁹DIVKLTIYDCIRRRRRRRRR (SEQ ID NO: 170) T41-R⁹DIVKLTVYDCLRRRRRRRRR (SEQ ID NO: 171) T42-R⁹DIVKLTVYDCVRRRRRRRRR (SEQ ID NO: 172) T43-R⁹DIDKLTEYDSIRRRRRRRRR (SEQ ID NO: 173) T44-R⁹DIPKLGVPDCIRRRRRRRRR (SEQ ID NO: 174) T45-R⁹ICDYVTLKVIDRRRRRRRRR (SEQ ID NO: 175) T46-R⁹VDLVIDCIYKTRRRRRRRRR (SEQ ID NO: 176) T47-R⁹DIVKLTVYDCIDIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 177) T48-R⁹IVKLTVYDCIRRRRRRRRR (N-succinyl) (SEQ ID NO: 178) T49-R⁹DIVKLTVYDCIGRRRRRRRRR (SEQ ID NO: 179) T50-R⁹DIVKLTVYDCIRRRRRRRRR (N-acetyl) (SEQ ID NO: 180) T51-R⁹AIVKLTVYACIRRRRRRRRR (SEQ ID NO: 181) T52-R⁹AIIKVYVYACIRRRRRRRRR (SEQ ID NO: 182) T53D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 183) T54D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg  (SEQ ID NO: 184)T55 D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile(SEQ ID NO: 185) T56Asp-Ile-Val-Lys-Leu-Thr-Val-Tyr-Asp-Cys-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ ID NO: 186)   1YIKVQKQDIVKLTVYDCISMIGLCA (SEQ ID NO: 187)   2IKVQKQDIVKLTVYDCISMIGLCA (SEQ ID NO: 188)   3KVQKQDIVKLTVYDCISMIGLCA (SEQ ID NO: 189)   4VQKQDIVKLTVYDCISMIGLCA (SEQ ID NO: 190)   5QKQDIVKLTVYDCISMIGLCA (SEQ ID NO: 191)   6KQDIVKLTVYDCISMIGLCA (SEQ ID NO: 192)   7QDIVKLTVYDCISMIGLCA (SEQ ID NO: 193)   8DIVKLTVYDCISMIGLCA (SEQ ID NO: 194)   9IVKLTVYDCISMIGLCA (SEQ ID NO: 195)  10 VKLTVYDCISMIGLCA (SEQ ID NO: 196) 11 KLTVYDCISMIGLCA (SEQ ID NO: 197)  12 LTVYDCISMIGLCA (SEQ ID NO: 198) 13 TVYDCISMIGLCA (SEQ ID NO: 199)  14 VYDCISMIGLCA (SEQ ID NO: 200)  15YDCISMIGLCA (SEQ ID NO: 201)  16 DCISMIGLCA (SEQ ID NO: 202)  17CISMIGLCA (SEQ ID NO: 203)  18 ISMIGLCA (SEQ ID NO: 204)  19SMIGLCA (SEQ ID NO: 205)  20 MIGLCA (SEQ ID NO: 206)  21IGLCA (SEQ ID NO: 207)  22 YIKVQKQDIVKLTVYDCISMIGLC (SEQ ID NO: 208)  23YIKVQKQDIVKLTVYDCISMIGL (SEQ ID NO: 209)  24YIKVQKQDIVKLTVYDCISMIG (SEQ ID NO: 210)  25YIKVQKQDIVKLTVYDCISMI (SEQ ID NO: 211)  26YIKVQKQDIVKLTVYDCISM (SEQ ID NO: 212)  27YIKVQKQDIVKLTVYDCIS (SEQ ID NO: 213)  28YIKVQKQDIVKLTVYDCI (SEQ ID NO: 214)  29YIKVQKQDIVKLTVYDC (SEQ ID NO: 215)  30 YIKVQKQDIVKLTVYD (SEQ ID NO: 216) 31 YIKVQKQDIVKLTVY (SEQ ID NO: 217)  32 YIKVQKQDIVKLTV (SEQ ID NO: 218) 33 YIKVQKQDIVKLT (SEQ ID NO: 219)  34 YIKVQKQDIVKL (SEQ ID NO: 220)  35YIKVQKQDIVK (SEQ ID NO: 221)  36 YIKVQKQDIV (SEQ ID NO: 222)  37YIKVQKQDI (SEQ ID NO: 223)  38 YIKVQKQD (SEQ ID NO: 224)  39YIKVQKQ (SEQ ID NO: 225)  40 YIKVQK (SEQ ID NO: 226)  41YIKVQ (SEQ ID NO: 227)  42 (S1)EMFTILEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 228)  43 (S2)MFTILEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 229)  44 (S3)FTILEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 230)  45TILEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 231)  46ILEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 232)  47LEEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 233)  48EEYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 234)  49EYFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 235)  50YFMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 236)  51FMYRGLLGLRIKYGRLFNEI (SEQ ID NO: 237)  52MYRGLLGLRIKYGRLFNEI (SEQ ID NO: 238)  53YRGLLGLRIKYGRLFNEI (SEQ ID NO: 239)  54RGLLGLRIKYGRLFNEI (SEQ ID NO: 240)  55 GLLGLRIKYGRLFNEI (SEQ ID NO: 241) 56 LLGLRIKYGRLFNEI (SEQ ID NO: 242)  57 LGLRIKYGRLFNEI (SEQ ID NO: 243) 58 GLRIKYGRLFNEI (SEQ ID NO: 244)  59 LRIKYGRLFNEI (SEQ ID NO: 245)  60RIKYGRLFNEI (SEQ ID NO: 246)  61 IKYGRLFNEI (SEQ ID NO: 247)  62KYGRLFNEI (SEQ ID NO: 248)  63 YGRLFNEI (SEQ ID NO: 249)  64GRLFNEI (SEQ ID NO: 250)  65 RLFNEI (SEQ ID NO: 251)  66LFNEI (SEQ ID NO: 252)  67 EMFTILEEYFMYRGLLGLRIKYGRLFNE (SEQ ID NO: 253) 68 (S4) EMFTILEEYFMYRGLLGLRIKYGRLFN (SEQ ID NO: 254)  69 (S5)EMFTILEEYFMYRGLLGLRIKYGRLF (SEQ ID NO: 255)  70 (S6)EMFTILEEYFMYRGLLGLRIKYGRL (SEQ ID NO: 256)  71 (S7)EMFTILEEYFMYRGLLGLRIKYG R (SEQ ID NO: 257)  72 (S8)EMFTILEEYFMYRGLLGLRIKYG (SEQ ID NO: 258)  73 (S9)EMFTILEEYFMYRGLLGLRIKY (SEQ ID NO: 259)  74 (S10)EMFTILEEYFMYRGLLGLRIK (SEQ ID NO: 260)  75 (S11)EMFTILEEYFMYRGLLGLRI (SEQ ID NO: 261)  76 (S12)EMFTILEEYFMYRGLLGL R (SEQ ID NO: 262)  77 (S13)EMFTILEEYFMYRGLLGL (SEQ ID NO: 263)  78EMFTILEEYFMYRGLLG (SEQ ID NO: 264)  79 (S14)EMFTILEEYFMYRGLL (SEQ ID NO: 265)  80 (S15)EMFTILEEYFMYRGL (SEQ ID NO: 266)  81 (S16)EMFTILEEYFMYRG (SEQ ID NO: 267)  82 EMFTILEEYFMY R (SEQ ID NO: 268) 83 (S17) EMFTILEEYFMY (SEQ ID NO: 269)  84 EMFTILEEYFM (SEQ ID NO: 270) 85 EMFTILEEYF (SEQ ID NO: 271)  86 EMFTILEEY (SEQ ID NO: 272)  87EMFTILEE (SEQ ID NO: 273)  88 EMFTILE (SEQ ID NO: 274)  89EMFTIL (SEQ ID NO: 275)  90 EMFTI (SEQ ID NO: 276)  91MFTILLEEYFMYRGLL (SEQ ID NO: 277)  92 FTILLEEYFMYRGLL (SEQ ID NO: 278) 93 TILLEEYFMYRGLL (SEQ ID NO: 279)  94 ILLEEYFMYRGLL (SEQ ID NO: 280) 95 LLEEYFMYRGLL (SEQ ID NO: 281)  96 LEEYFMYRGLL (SEQ ID NO: 282)  97EYFMYRGLL (SEQ ID NO: 283)  98 YFMYRGLL (SEQ ID NO: 284)  99FMYRGLL (SEQ ID NO: 285) 100 MYRGLL (SEQ ID NO: 286) 101YRGLL (SEQ ID NO: 287) 102 (S18) MFTILEEYFMYRGLLGLRI (SEQ ID NO: 288)103 (S19) FTILLEEYFMYRGLLGLRI (SEQ ID NO: 289) 104 (S20)TILLEEYFMYRGLLGLRI (SEQ ID NO: 290) 105 (S21)ILLEEYFMYRGLLGLRI (SEQ ID NO: 291) 106 (S22)LLEEYFMYGLLGLRI (SEQ ID NO: 292) 107 EYFMYRGLLGLRIKYG (SEQ ID NO: 293)108 YFMYRGLLGLRIKYG (SEQ ID NO: 294) 109 FMYRGLLGLRIKYG (SEQ ID NO: 295)110 MYRGLLGLRIKYG (SEQ ID NO: 296) 111 YRGLLGLRIKYG (SEQ ID NO: 297) 112RGLLGLRIKYG (SEQ ID NO: 298) 113 GLLGLRIKYG (SEQ ID NO: 299) 114 LLGLRIKYG (SEQ ID NO: 300) 115 LGLRIKYG (SEQ ID NO: 301) 116GLRIKYG (SEQ ID NO: 302) 117 LRIKYG (SEQ ID NO: 303) 118RIKYG (SEQ ID NO: 304) 119 EEYFM (SEQ ID NO: 305) 120EEYFMY (SEQ ID NO: 306) 121 EEYFMY R (SEQ ID NO: 307) 122EEYFMYRG (SEQ ID NO: 308) 123 EEYFMYRGL (SEQ ID NO: 309) 124EEYFMYRGLL (SEQ ID NO: 310) 125 EEYFMYRGLLG (SEQ ID NO: 311) 126EEYFMYRGLLGL (SEQ ID NO: 312) 127 EEYFMYRGLLGL R (SEQ ID NO: 313) 128EEYFMYRGLLGLRI (SEQ ID NO: 314) 129 EEYFMYRGLLGLRIK (SEQ ID NO: 315) 130EEYFMYRGLLGLRIKY (SEQ ID NO: 316)  T1 IVKLTVYDCI (SEQ ID NO: 317)  T2DIVKLTVYDC (SEQ ID NO: 318)  T3 AIVKLTVYDCI (SEQ ID NO: 319)  T4DAVKLTVYDCI (SEQ ID NO: 320)  T5 DIAKLTVYDCI (SEQ ID NO: 321)  T6DIVALTVYDCI (SEQ ID NO: 322)  T7 DIVKATVYDCI (SEQ ID NO: 323)  T8DIVKLAVYDCI (SEQ ID NO: 324)  T9 DIVKLTAYDCI (SEQ ID NO: 325) T10DIVKLTVADCI (SEQ ID NO: 326) T11 DIVKLTVYACI (SEQ ID NO: 327) T12DIVKLTVYDAI (SEQ ID NO: 328) T13 DIVKLTVYDCA (SEQ ID NO: 329) T14EIVKLTVYDCI (SEQ ID NO: 330) T15 DIVKLTVYECI (SEQ ID NO: 331) T16DIVRLTVYDCI (SEQ ID NO: 332) T17 DIVHLTVYDCI (SEQ ID NO: 333) T18KIVKLTVYKCI (SEQ ID NO: 334) T19 DIVELTVYDCI (SEQ ID NO: 335) T20DIVKLSVYDCI (SEQ ID NO: 336) T21 DIVKLYVYDCI (SEQ ID NO: 337) T22DIVKLTVSDCI (SEQ ID NO: 338) T23 DIVKLTVTDCI (SEQ ID NO: 339) T24DIVKLTVWDCI (SEQ ID NO: 340) T25 DIVKLTVFDCI (SEQ ID NO: 341) T26DIVKLTVYDMI (SEQ ID NO: 342) T27 DIVKLTVYDSI (SEQ ID NO: 343) T28DIVKLTVYDXaaI (SEQ ID NO: 344) T29 DIVKLTVYDXaaI (SEQ ID NO: 345) T30DIVKLTVYDXaaI (SEQ ID NO: 346) T31 DIVKLTVYDXaaI (SEQ ID NO: 347) T32DIVKLTVYDXaaI (SEQ ID NO: 348) T33 DLVKLTVYDCI (SEQ ID NO: 349) T34DVVKLTVYDCI (SEQ ID NO: 350) T35 DILKLTVYDCI (SEQ ID NO: 351) T36DIIKLTVYDCI (SEQ ID NO: 352) T37 DIVKVTVYDCI (SEQ ID NO: 353) T38DIVKITVYDCI (SEQ ID NO: 354) T39 DIVKLTLYDCI (SEQ ID NO: 355) T40DIVKLTIYDCI (SEQ ID NO: 356) T41 DIVKLTVYDCL (SEQ ID NO: 357) T42DIVKLTVYDCV (SEQ ID NO: 358) T43 DIDKLTEYDSI (SEQ ID NO: 359) T44DIPKLGVPDCI (SEQ ID NO: 360) T45 ICDYVTLKVID (SEQ ID NO: 361) T46VDLVIDCIYKT (SEQ ID NO: 362) T47 DIVKLTVYDCIDIVKLTVYDCI (SEQ ID NO: 363)T48 IVKLTVYDCI (N-succinyl) (SEQ ID NO: 364) T49DIVKLTVYDCIG (SEQ ID NO: 365) T50DIVKLTVYDCI (N-acetyl) (SEQ ID NO: 366) T51 AIVKLTVYACI (SEQ ID NO: 367)T52 AIIKVYVYACI (SEQ ID NO: 368) Legend to Table 1: Xaa in peptide T31and T31-R⁹ is α-Aminobutyric acid; Xaa in peptide T32 and T32-R⁹ isL-Norvaline; Xaa in peptide T30 and T30-R⁹ is L-Cysteine(S-Acm); Xaa inpeptide T28 and T28-R⁹ is L-Cysteine(S-carboxymethyl); and Xaa inpeptide T29 and T29-R⁹ is L-Cysteine(S-carbamidomethyl).

Peptides of the invention and compositions comprising the same areeffective to modulate cellular activity. In some embodiments, modulatingcellular activity is accomplished by modulating cellular signaling.Activities can be regulated, for example, by a toll-like receptor. Insome embodiments, peptides and compositions of the invention inhibitcytokine secretion. In some embodiments, peptides and compositions ofthe invention enhance cytokine secretion. In some embodiments, cytokinesecretion is in response to toll-like receptor-dependent stimulation.

In some embodiments, administration of a peptide derived from A52R orP13 can inhibit cytokine secretion by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80% or 90%. In some embodiments, administration of a peptidederived from A52R or P13 can enhance cytokine secretion by 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments the cytokinesecretion is a result of TLR-dependent signaling.

In some embodiments, the activity mediated by a toll-like receptor isTNF-α secretion. Peptides and compositions of the invention areeffective to provide 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by LPS. Peptides andcompositions of the invention are effective to provide 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or 90% inhibition of TNF-α secretion followingstimulation by CpG-ODN. Peptides and compositions of the invention areeffective to provide 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by LPS and/orCpG-ODN. Peptides and compositions of the invention are effective toprovide 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% enhancement ofTNF-α secretion following stimulation by LPS. Peptides and compositionsof the invention are effective to provide 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90% enhancement of TNF-α secretion following stimulation byCpG-ODN. Peptides and compositions of the invention are effective toprovide 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% enhancement ofTNF-α secretion following stimulation by LPS and/or CpG-ODN.

Some embodiments of the invention contemplate a peptide comprising theamino acid sequence DIVKLTVYDCI (SEQ ID NO: 369), linked to a 9-arginine(SEQ ID NO: 379) cell transduction sequence or other type of celltransduction sequence. The peptide DIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 378)effectively inhibits cytokine secretion in response to TLR activation.The peptide had no effect on cytokine secretion resulting from cellactivation that was initiated independent of TLR stimulation. Employinga mouse model of otitis media with effusion (OME), administration ofheat-inactivated Streptococcus pneumoniae (S. pneumoniae) into themiddle ears of BALB/c mice resulted in a significant inflammatoryresponse that was dramatically reduced with peptide treatment.Experiments have also demonstrated that the peptide will reducepro-inflammatory mediators in a mouse model of LPS-induced septic shock.The peptide is effective in the treatment of chronic inflammationinitiated by bacterial or viral infections.

In some embodiments, the invention contemplates a pharmaceuticalcomposition effective to:

a) decrease the amount of an middle ear fluid by about 30-80%;

b) decrease infiltrating cell number in middle ear fluid by about30-80%; and

c) decrease the thickness of a tympanic membrane by about 30-80% in amouse comprising inflammation of the ear, wherein the composition isadministered to the mouse in an amount of about 0.1 μg to 60 μg. In someembodiments, the pharmaceutical composition is an aural pharmaceuticalcomposition. In some embodiments, the pharmaceutical composition isformulated for intra-aural administration. In some embodiments, thepharmaceutical composition is formulated as a drop. In some embodiments,the pharmaceutical composition is formulated as an ear drop.

In some embodiments, a pharmaceutical composition of the inventioncomprises a peptide. In some embodiments, the peptide is P13 or avariant, derivative, stereoisomer, or analogue thereof. In someembodiments, the peptide is P13, a peptide comprising P13 and apoly-argenine domain, or a peptide of Table 1. In some embodiments, thepeptide is P13. In some embodiments, the peptide is derived from A52R.In some embodiments, the peptide is a peptide of S1-S22. The inventionalso contemplates a method of treating ear inflammation in an animal inneed or want thereof, the method comprising administering to the animala pharmaceutical composition of the invention.

The in vivo effectiveness of P13 was demonstrated using a mouse model ofotitis media (OM). OM is an inflammatory disease of the middle earaccompanied by fluid accumulation. It is characterized by aninfiltration of leukocytes, macrophages, and mast cells and a release ofinflammatory mediators and enzymes (Reference 21). These mediatorsincrease vascular permeability and secretory activity, and initiate acascade of inflammatory events, resulting in fluid accumulation andmucin secretion (References 26 and 27). The initiation of inflammationin OM has been attributed to a variety of factors, including bacterialor viral infections, Eustachian tube dysfunction, and allergy. However,the evidence points to a bacterial etiology leading to cytokineactivation in the majority of cases. Bacteria have been cultured from upto 40% of effusions and studies have shown bacterial DNA by PCR inapproximately 80% of effusions, often in the absence of viable organismsin culture (Reference 28). The most common bacteria invading the middleear are S. pneumoniae, H. influenzae, and M. catarralis. These threebacteria account for 85% of acute middle ear infections (Reference 27),with S. pneumoniae being the most frequent cause. Initially, livebacteria trigger acute inflammation, which is designed to eliminate thepathogen. During acute infection, interference with the innate immuneresponse would be potentially harmful to the host and can lead tofurther bacterial spread. Acute inflammation initiated by bacterialinfections self-resolves or is treatable by antibiotics. Chronicinflammation involves continued activation of the immune system, oftenby non-viable bacterial products. OM is often prolonged or anti-bioticresistant, suggesting TLR stimulation in the absence of live bacteria.

Treatment of mice with P13 in the experiments disclosed herein resultedin a significant reduction in bacterial-induced inflammation in themiddle ear. Fluid accumulation, infiltrating cells, and tympanicmembrane thickness in the middle ear were all dramatically reduced withpeptide treatment. Administration of heat-inactivated bacteria, whichhave a number of potential TLR ligands, induced an inflammatory responsein the middle ear most likely resulting from activation of multipleTLRs. The use of heat-inactivated bacteria allowed for an examination ofpeptide inhibition of inflammation without the potential for thebacterial spread that can occur in an acute infection initiated withlive bacteria. The ability of peptide P13 to inhibit this responsesignificantly in vivo is consistent with the in vitro data showinginhibition of cytokine secretion in response to multiple TLR ligandsused either individually or in combination. In these studies, a singledose of peptide was administered at the same time as heat-inactivated S.pneumoniae into the middle ears of normal BALB/c mice. The in vitro dataalso showed inhibition of cytokine secretion even when peptide P13 wasadded several hours after initiation of TLR activation.

In addition to P13 and the peptides of Table 1, the invention disclosedherein also contemplates peptides more broadly characterized asvariants, derivatives, stereoisomers, or analogues of any peptide of theinstant invention. These terms are not exclusive, and can becontemplated in concert to describe peptides that are described by oneor more of the terms. Many such peptides, not all of which are expresslydisclosed herein, are contemplated as peptides, as components of aformulation or pharmaceutical composition, and as elements of themethods and uses of the instant invention.

In some embodiments, the variant, derivative, stereoisomer, or analogueof a peptide of the invention comprises a peptide comprising a peptideof the invention and a poly-argenine sequence. In some embodiments, thevariant, derivative, stereoisomer, or analogue of a peptide of theinvention comprises one or more D-amino acid residues. In someembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of theamino acid residues of the variant, derivative, stereoisomer, oranalogue of a peptide of the invention possess the D-configuration. Insome embodiments, all the amino acid residues of the variant,derivative, stereoisomer, or analogue of a peptide of the inventionpossess the D-configuration.

In some embodiments, the variant, derivative, stereoisomer, or analogueof a peptide of the invention comprises one or more D-amino acidresidues in the region of the peptide analogous to or derived from apeptide of the invention. In some embodiments, the region of the peptideanalogous to or derived from P13 comprises more than one D-amino acidresidue.

In some embodiments, the region of the peptide analogous to or derivedfrom a peptide of the invention comprises a D-amino acid residue. Insome embodiments, the poly-argenine sequence comprises a D-amino acidresidue. In some embodiments, the poly-argenine sequence comprises morethan one D-amino acid residue. In some embodiments, either the region ofthe peptide analogous to or derived from a peptide of the invention orthe poly-argenine sequence comprises a D-amino acid residue. In someembodiments, either the region of the peptide analogous to or derivedfrom a peptide of the invention or the poly-argenine sequence comprisesmore than one D-amino acid residue. In some embodiments, both the regionof the peptide analogous to or derived from a peptide of the inventionand the poly-argenine sequence independently comprise a D-amino acidresidue. In some embodiments, both the region of the peptide analogousto or derived from a peptide of the invention and the poly-argeninesequence independently comprise more than one D-amino acid residue.

In some embodiments comprising a peptide comprising a variant,derivative, stereoisomer, or analogue of a peptide of the inventioncomprising a poly-argenine sequence, the region of the peptide analogousto or derived from P13 consists of D-amino acid residues. In someembodiments, the region of the peptide analogous to or derived from apeptide of the invention consists of D-amino acid residues, and thepoly-argenine sequence consists of L-amino acid residues. In someembodiments, the region of the peptide analogous to or derived from apeptide of the invention consists of D-amino acid residues, and thepoly-argenine sequence comprises one or more D-amino acid residues.

In some embodiments, the poly-argenine sequence consists of D-amino acidresidues. In some embodiments, the poly-argenine sequence consists ofD-amino acid residues, and the region of the peptide analogous to orderived from a peptide of the invention consists of L-amino acidresidues. In some embodiments, the poly-argenine sequence consists ofD-amino acid residues, and the region of the peptide analogous to orderived from a peptide of the invention comprises one or more D-aminoacid residues.

In some embodiments comprising a peptide comprising a variant,derivative, stereoisomer, or analogue of a peptide of the inventioncomprising a poly-argenine sequence, the peptide consists of D-aminoacid residues.

In some embodiments, a peptide comprises an amino acid residuecomprising a stereogenic side chain, for example, threonine,allo-threonine, isoleucine, or allo-isoleucine. In some embodiments, apeptide comprises a D-allo-threonine, or D-allo-isoleucine residue.

Diseases

The initiation of an inflammatory response to pathogens is a componentof the innate immune response and is designed to control infection.However, the sustained production of inflammatory mediators can lead tochronic inflammation, tissue damage and disease development. Thesignaling cascade initiated by PAMP/TLR interactions and culminating incell activation has been associated with many disease states, includingotitis media, inner ear inflammation, sepsis, autoimmune diseases,asthma, heart disease and cancer (Reference 29). OM is an inflammatorydisease of the middle ear. OM is often prolonged or antibioticresistant; these characteristics suggest TLR stimulation in the absenceof live bacteria. An abnormal TLR signaling response could lead toexaggerated cell-activation responses contributing to sepsis (Reference30 and 31).

Inflammation is also an aspect of autoimmunity, and is hypothesized toplay a role in tissue destruction in diseases such as multiplesclerosis, rheumatoid arthritis and insulin-dependent diabetes mellitus(Reference 32). Cells of the innate immune system have an essential rolein acquired/adaptive immunity. TLR proteins are involved in thematuration and activation of dendritic cells, the antigen-presentingcell type considered most relevant to development of acquired immunity(Reference 33). Allergic asthma is an example of a chronic inflammatorydisease with an adaptive immune response, and the TLR signaling pathwayis implicated in the induction phase of an allergic phenotype (Reference30). Bacterial and viral infections, causing increased inflammatory cellactivation, are the main cause of exacerbations in diseases such asasthma and COPD (chronic obstructive pulmonary disease) (Reference 30).

In some embodiments, the inflammation is caused by a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof. In someembodiments, the inflammation is associated with a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof. In someembodiments, the inflammation is correlated with a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof. In someembodiments, the inflammation is accompanied by a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof. In someembodiments, an organism having the inflammation also has a virus,bacteria, fungi, antigen, self-antigen, or a combination thereof.

In some embodiments, the peptides provided herein are administered to asubject for the treatment of inflammation. Non-limiting examples of thecauses of inflammation include viral, bacterial or fungal infection. Insome embodiments, the inflammation is a result of a response to aself-antigen or any other antigen. In some embodiments, the inflammationis a result of a response to an anti-self-antigen

In some embodiments, the inflammation comprises inflammation of the ear.The inflammation of the ear can be inflammation of the inner ear and/ormiddle ear. In some embodiments, the inflammation comprises otitismedia. In some embodiments, pharmaceutical compositions of the instantinvention are used for the treatment of otitis media.

C3H/HeJ mice, defective in TLR4 signaling, are known to develop chronicotitis media (“COM”) spontaneously. Such mice with inner earinflammation may show sensorineural hearing loss in addition to middleear conductive hearing loss. Histologic examination of the middle andinner ear of C3H/HeJ mice with COM has documented a thickening of themucosal surfaces of the middle ear, thickening of the round windowmembrane, fibrosis, labyrinthitis, and Eustachian tube obstruction.These histologic changes correlate with the histology seen with humantemporal bone from patients with a history of COM and labyrinthitis. Themouse studies have shown that C3H/HeJ mice with COM have gram-negativeKlebsiella bacteria in the middle ear. The analogies between theobservations in the mouse experiments and those in human COM andlabrinthitis patients suggest that mouse experiments with a peptide orpharmaceutical composition of the invention disclosed herein provideresults predictive of what the therapeutic effect would be in a humansubject.

In some embodiments, otitis media is associated with hearing loss orreduced hearing. Administration of a peptide of the invention of apharmaceutical composition thereof can improve the hearing of theaffected organism back to ordinary hearing levels. In some embodiments,administration of a therapeutically-effective amount of a pharmaceuticalcomposition to an organism in need or want thereof improves the hearingof the organism, wherein the organism has both otitis media and lessenedhearing. In some embodiments, the peptide or pharmaceutical compositionis administered topically. In some embodiments, upon administration of apeptide of the invention of a pharmaceutical composition thereof, anorganism with hearing loss recovers hearing faster than hearing would berecovered without administration of the peptide or pharmaceuticalcomposition.

In some embodiments, administration of a therapeutically-effectiveamount of a peptide of the invention or a pharmaceutical compositioncomprising the same to an organism with otitis media, wherein theorganism is in need or want thereof, provides therapeutic relief of asymptom of otitis media. Non-limiting examples of the symptoms of otitismedia include otalgia (pain), otorrhea, fever, irritability, anorexia,vomiting or diarrhea. In some embodiments, the symptom is pain.

In some embodiments, the inflammation comprises inflammation of theskin, joints, muscular tissue, brain, or connective tissue. In someembodiments, pharmaceutical compositions of the instant invention areused for the treatment of inflammation of the skin, joints, musculartissue, brain, or connective tissue.

In some embodiments, the inflammation comprises arthritis, dermatitis,Lupus erythematosus, meningitis, or psoriasis. In some embodiments, thearthritis comprises osteoarthritis, rheumatoid arthritis, septicarthritis, gout, pseudo-gout, juvenile idiopathic arthritis, Still'sdisease, or ankylosing spondylitis. In some embodiments, the dermatitiscomprises spongiotic dermatitis, childhood eczema, allergic contactdermatitis, seborrhoeic dermatitis, dyshidrotic dermatitis, urticaria,vesicular or bullous dermatitis, or papular urticaria. In someembodiments, the psoriasis comprises plaque psoriasis, flexuralpsoriasis, guttate psoriasis, pustular psoriasis, nail psoriasis,psoriatic arthritis, or erythrodermic psoriasis.

In some embodiments, the administration comprises topical application.In some embodiments, the administration comprises topical application tothe skin, hair, outer ear, tympanic membrane, nasal cavity, buccalcavity, or sublingual cavity.

In some embodiments, the peptides of the invention and pharmaceuticalcomposition comprising the same are effective for the treatment ofsinusitis. In some embodiments, administration of a pharmaceuticalcomposition of the invention to an organism in need or want thereofprovides a therapeutic effect on sinusitis or a symptom thereof.Non-limiting examples of symptoms of sinusitis include stuffy or runnynose, nasal discharge, bloody nasal discharge, sneezing, coughing, nasalpain, headache, postnasal drip, itchy face, diminished scent or tastesenses, bad breath, fever, chill, dental pain, or face pain. In someembodiments, a pharmaceutical composition of the invention isadministered as an aerosol, vapor, spray, or mist.

Therapeutic Uses

A, “patient,” “subject,” or “host,” to be treated with a pharmaceuticalcomposition of the present invention may mean either a human ornon-human animal. In some embodiments, the subject is human. Thepeptides of the present invention are useful in the treatment of suchdiseases and disorders such as but not limited to those involvinginflammation. In one embodiment, the peptides and pharmaceuticalcompositions of the present invention may be used in the manufacture ofa medicament for any number of uses, including, for example, treatingany disease or other treatable condition of a patient.

A, “therapeutic effect,” as the term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderbeing treated. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the patient, notwithstanding that the patient may still beafflicted with the underlying disorder. For prophylactic benefit, thecompositions may be administered to a patient at risk of developing aparticular disease, or to a patient reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease may not have been made. A prophylactic effect includes delayingor eliminating the appearance of a disease or condition, delaying oreliminating the onset of symptoms of a disease or condition, slowing,halting, or reversing the progression of a disease or condition, or anycombination thereof.

Administration

A pharmaceutical composition containing a peptide can be administered topatients along with pharmaceutical excipients or diluents. Non-limitingexamples of suitable pharmaceutical excipients or diluents includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium carbonate, magnesium stearate, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, buffered water, phosphate bufferedsaline and the like. These compositions can take the form of drops,solutions, suspensions, tablets, pills, capsules, powders,sustained-release formulations and the like. In some embodiments, thecomposition is an ear drop. In another preferred embodiment thecomposition containing a peptide in any form could be further modulatedusing suitable excipients and diluents including lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearateand mineral oil. The formulations can additionally include lubricatingagents, wetting agents, emulsifying and suspending agents, preservingagents, sweetening agents or flavoring agents. The compositions can beformulated in a unit dosage form, each dosage containing, for example,from about 1 ng to 1000 mg of the peptide. In some embodiments, a dosecontains from 100-1000 mg of the peptide. In some embodiments, a dosecontains from 100-500 mg of the peptide. In some embodiments, a dosecontains from 200-300 mg of the peptide The term, “unit dosage form,”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticaldiluents or excipients. These may be administered to humans, domesticpets, livestock, or other animals with a pharmaceutically-acceptablediluents or excipients, in unit dosage form. Administration may betopical, intraaural, parenteral, intravenous, intra-arterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, by suppositories, or oraladministration. Formulations for oral use include tablets containing theactive ingredient(s) in a mixture with non-toxicpharmaceutically-acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose and sorbitol),lubricating agents, glidants, and antiadhesives (e.g., magnesiumstearate, zinc stearate, stearic acid, silicas, hydrogenated vegetableoils, or talc).

The active therapeutic formulation of the invention can be provided inlyophilized form for reconstituting, for instance, in isotonic, aqueous,or saline buffers for parental, subcutaneous, intradermal, intramuscularor intravenous administration. The subject composition of the inventionmay also be administered to the patient in need of a therapeutic peptideby liquid preparations for orifice, e.g. oral, intraaural, nasal, orsublingual, administration such as suspensions, syrups or elixirs. Thesubject composition of the invention may also be prepared for oraladministration such as capsules, tablets, pills, and the like, as wellas chewable solid formulations. The subject composition of the inventionmay also be prepared as a cream for dermal administration such asliquid, viscous liquid, paste, or powder. The subject composition of theinvention may also be prepared as powder for lung administration with orwithout aerosolizing component. The composition of the invention can beprepared as a drop, for example, an ear drop.

The presently disclosed compositions can be used for delivery in oral,intraaural, intranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular and ocular forms aswell as being able to traverse the blood-brain barrier.

Dosages

The dosage of any peptide of the present invention will vary dependingon the symptoms, age and body weight of the patient, the nature andseverity of the disorder to be treated or prevented, the route ofadministration, and the form of the composition. Any of the subjectformulations may be administered in a single dose or in divided doses.Dosages for the peptides of the present invention may be readilydetermined by techniques known to those of skill in the art or as taughtherein. Also, the present invention contemplates mixtures of more thanone subject peptide, as well as other therapeutic agents.

In certain embodiments, the dosage of the subject peptide will generallybe in the range of about 0.01 ng to about 10 g per kg body weight,specifically in the range of about 1 ng to about 0.1 g per kg, and morespecifically in the range of about 100 ng to about 10 mg per kg.

An effective dose or amount, and any possible affects on the timing ofadministration of the formulation, may need to be identified for anyparticular peptide of the present invention. This may be accomplished byroutine experiment as described herein, using one or more groups ofanimals, or in human trials if appropriate. The effectiveness of anypeptide and method of treatment or prevention may be assessed byadministering the supplement and assessing the effect of theadministration by measuring one or more indices associated with theneoplasm of interest, and comparing the post-treatment values of theseindices to the values of the same indices prior to treatment.

The precise time of administration and amount of any particular peptidethat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular peptide, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like. The guidelines presented herein may beused to optimize the treatment, e.g., determining the optimum timeand/or amount of administration, which will require no more than routineexperimentation consisting of monitoring the subject and adjusting thedosage and/or timing.

While the subject is being treated, the health of the subject may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. Treatment, includingsupplement, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters, the first such reevaluation typicallyoccurring at the end of four weeks from the onset of therapy, andsubsequent reevaluations occurring every four to eight weeks duringtherapy and then every three months thereafter. Therapy may continue forseveral months or even years, with a minimum of one month being atypical length of therapy for humans. Adjustments to the amount(s) ofpeptide administered and possibly to the time of administration may bemade based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the peptide. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The combined use of several peptides of the present invention, oralternatively other peptides, may reduce the required dosage for anyindividual component because the onset and duration of effect of thedifferent components may be complimentary. In such combined therapy, thedifferent peptides may be delivered together or separately, andsimultaneously or at different times within the day.

Toxicity and therapeutic efficacy of subject peptides may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ and the ED₅₀. Although peptidesthat exhibit toxic side effects may be used, care should be taken todesign a delivery system that targets the peptides to the desired sitein order to reduce side effects.

The data obtained from cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany supplement, or alternatively of any components therein, liespreferably within a range of circulating concentrations that include theED₅₀, with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For peptides of the present invention, the therapeuticallyeffective dose may be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test peptide which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information may be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography (HPLC).

Formulations

The peptide-based compositions of the present invention may beadministered by various means, depending on their intended use, as iswell known in the art. For example, if compositions of the presentinvention are to be administered orally, they may be formulated astablets, capsules, granules, powders or syrups. Alternatively,formulations of the present invention may be administered parenterallyas injections (intravenous, intramuscular or subcutaneous), dropinfusion preparations or suppositories. The peptide-based compositionscan also be administered into deep lung by aerosolizing the compositioninto 1-5 um particle using standard techniques known in the art eitherwith or without addition of aerosolizing excipient. For application bythe ophthalmic mucous membrane route, compositions of the presentinvention may be formulated as eye drops or eye ointments. Auralpharmaceutical compositions can be formulated as ear drops, ointments,creams, liquids, gels, or salves for application to the ear, eitherinternally or superficially. These formulations may be prepared byconventional means, and, if desired, the compositions may be mixed withany conventional additive, such as an excipient, a binder, adisintegrating agent, a lubricant, a solubilizing agent, a suspensionaid, an emulsifying agent or a coating agent.

In formulations of the subject invention, wetting agents, emulsifiersand lubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants may bepresent in the formulated agents.

Subject peptide-based compositions may be suitable for oral, intraaural,nasal, topical (including buccal and sublingual), rectal, vaginal,aerosol and/or parenteral administration. The formulations of thepeptide-based compositions may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. The amounts of composition that may be combined with otherexcipients to produce a single dose may vary depending upon the subjectbeing treated, and the particular mode of administration.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition thereof as an active ingredient. Compositions of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject peptidecomposition is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepeptide compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, gels, solutions,suspensions, syrups and elixirs. The liquid dosage peptide formulationmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspension dosage of the peptide formulation may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The peptide formulations for rectal or vaginal administration may bepresented as a suppository, which may be prepared by mixing a peptidewith one or more suitable carriers and other excipients comprising, forexample, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and releasepeptide. Formulations which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing excipients as are known in the art to beappropriate.

The peptide dosage formulations for transdermal administration of asubject composition includes drops, powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecomponent may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to asubject composition, excipients, such as animal and vegetable fats,oils, waxes, paraffin, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonite, silicic acid, talc and zincoxide, or mixtures thereof. The peptide compositions of the presentinvention may also be in the form of baby wipes.

Powders and sprays may contain, in addition to a subject composition,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays may additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The peptide compositions of the present invention may alternatively beadministered by aerosol. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containing thecompound. A non-aqueous (e.g., fluorocarbon propellant) suspension couldbe used. Sonic nebulizers may be used because they minimize exposing thepeptide to shear, which may result in degradation of the peptidescontained in the subject compositions.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of a subject composition together withconventional pharmaceutically-acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularsubject composition, but typically include non-ionic surfactants(Tweens, Pluronics, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a subject composition in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity may be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

EXAMPLES Example 1 Materials and Methods

Peptide of the Natural Configuration:

Peptides were synthesized by Mimotopes using their unique proprietaryparallel array synthesis platform. Each peptide contains an amino acidsequence of the invention and optionally a 9-residue arginine (SEQ IDNO: 379) cell transduction sequence positioned at the C-terminus of thepeptide to facilitate cellular uptake. The peptides are presented inTable 1.

Peptides with D-Amino Acid Residues are Synthesized as Peptides withL-Amino Acid Residues are Synthesized.

The D-amino acids with the appropriate protecting groups arecommercially available for use on automated systems to constructpeptides. Peptides described herein can be made entirely from L-aminoacids, entirely from D-amino acids, or from a mixture of both in anyproportion. A peptide is considered stereochemically-mixed if thepeptide comprises at least one L-, and at least one D-amino acidresidue. The transduction tag can similarly be made entirely fromL-amino acids, entirely from D-amino acids, or from a mixture of both inany proportion. Peptides differing only in stereochemistry may possessdiffering properties, benefits, specificities, affinities, andactivities at the same or at different receptors. In some cases,peptides differing only in stereochemistry may exhibit comparablespecificities and activities.

Each peptide is constructed both with and without a FITC-label(Fluorescein isothiocyanate). FITC labeled peptides are used for FACSanalysis. The peptides lacking the FITC label are used for in vitroinhibition assays and in vivo treatment studies.

Reagents:

Nuclease-resistant phosphorylated oligonucleotide was purchased fromOligos Etc., Inc. The sequence was 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ IDNO: 377) (CpG-oligodeoxynucleotide (ODN). TNF-α assays were performedusing assay kits purchased from R&D Systems. Mouse IL-1α and TNF-α werepurchased from R&D Systems. PMA (phorbol myristate acetate) and LPS(Lipopolysaccaride) were purchased from Sigma. The TLR ligandsflagellin, zymosan, and Poly (I:C) were purchased from Invivogen.Cytokine assays were performed using assay kits purchased from R&DSystems. (Heat-inactivated S. pneumoniae was the kind gift of Dr. ThomasDeMaria, The Ohio State University College of Medicine, Department ofOtolaryngology, Columbus, Ohio.)

Cell Lines and Cultures:

RAW264.7 (murine monocyte/macrophage cells (American Type CultureCollection) were cultured at 37° C. in a 5% CO₂ humidified incubator andgrown in DMEM (Invitrogen Life Technologies) supplemented with 10% (v/v)heat-inactivated FCS, 1.5 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin.

Cytokine Secretion:

RAW264.7 cells were plated at 1.5×10⁵ cells/well-3×10⁵ cells/well in48-well plates. After 24 hours, the cells were incubated with peptidesat various concentrations at room temperature in triplicate eitherbefore, simultaneous with, or after activation with various PAMPs for 18hours. Cell-free supernatants were analyzed for cytokines by ELISA, inquadruplicate. RAW264.7 cells were stimulated with either CpG-ODN (1μg/ml or 1.25 μg/ml), LPS (about 1 ng/ml), Poly (I:C) (about 10 μg/ml),flagellin (about 5 ng/ml), or zymosan (about 10 μg/ml). Dose responsecurves were done with each PAMP to determine optimal stimulationconcentration. For CpG-ODN stimulation, cells were incubated for 4 hoursat 37° C., supernatants collected, and TNF-α measured by ELISA. Anycytokine involved in inflammatory disease mediated toll receptorsignaling can be measured using similar assays.

Flow Cytometry:

Cells are analyzed by flow cytometry (FACScan, Becton Dickinson) usingCellquest software to quantify internalization of peptides. Gates aredrawn to exclude dead cells based on 7-AAD (7-amino-actinomycin D)staining Flourescence due to cell-surface binding of FITC-labeledpeptides is quenched using trypan blue. Data obtained are geometric meanfluorescent units (F) with background autofluorescence subtracted.

Immunoblotting:

RAW264.7 cells (6×10⁵) are plated in 12 well plates overnight. Cells areincubated for 15 minutes at room temperature with peptides to be testedor control scrambled peptides, and then stimulated with medium or LPS (1ng/ml) for either 15 or 30 minutes. Cells are lysed, and proteinsfractionated by SDS/PAGE (Sodium Dodecyl Sulfate/Polyacrylamide GelElectrophoresis) (12%). Immunoblotting is done using Phospho-IkB-α(Ser32) antibody (Cell Signaling), detected using horseradishperoxidase-conjugated secondary antibody, and visualized bychemiluminescence. Measurements of band intensity are made using theNucleo Tech Gel Expert software linked to an Epson expression 636scanner and expressed as intensity/area.

Cell Viability:

Cells were assayed for viability using CellTiter 96 Aq_(ueous) OneSolution Cell Proliferation Assay (Promega) following manufacturer'sinstructions. Briefly, cells were seeded in 96 well plates and incubatedovernight at 37° C. in a humidified 5% CO₂ atmosphere. When samples wereready to be assayed, 20 μl of reagent was added into each well, andincubated for 1.5 hrs at 37° C. in a humidified 5% CO₂ atmosphere.Absorbances were read at 490 nm using an ELx800 absorbance microplatereader (BioTek) and data analyzed with GenS software (BioTek). Somepeptides were evaluated for their effect on cell viability by trypanblue exclusion staining over a range of concentrations and then eachpeptide was tested for cytokine inhibition at the maximum concentrationthat had no effect on cell viability.

Example 2 Effect of Peptides on CpG-ODN-Induced Cytokine Secretion

The effect of several peptides on TNF-α secretion from RAW264.7 cells inresponse to stimulation by CpG-ODN was studied. Each peptide wasinitially tested at 3 concentrations, 37 μM, 22.2 μM, and 11.1 μM.Seventeen peptides demonstrating inhibition of TNF-α secretion of50-100% across all three doses are presented (Table 2). The peptideswere then tested at 11.1 μM, 7.4 μM, and 3.7 μM (Table 3). The peptidescontinued to demonstrate inhibitory activity at 7.4 μM, and twelve ofthe peptides had inhibitory activity at 3.7 μM. Cell viability wasexamined for each peptide at the concentrations tested for TNF-αinhibition. No decrease in cell viability was demonstrated.

TABLE 2  Percent Inhibition of TNF-αsecretion by S1-S22 peptides with a R⁹ sequence(SEQ ID NO: 379). RAW264.7 cells were plated at 3X10⁵ cells/well in 48-well plates.After 24 h the cells were incubated with peptide at various concentrations at roomtemperature in triplicate for 15 minutes and then stimulated with 1 ug/ml Cpg-ODN. Cells were then incubated for 4 hours at 37°C., supernatants collected, and TNF-αmeasured by ELISA. Percent inhibition was calculated by comparing TNF-αsecretionfrom cells incubated with peptide to control cells with no peptide treatment.Peptide 37 μM 22.2 μM 11.1 μM Sequence  42 (S1)-R9 97 96 85EMFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 42)  43 (S2)-R9 10094 91 MFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 43)  44 (S3)-R995 96 91 FTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 44) 68 (S4)-R9 100 100 95 EMFTILEEYFMYRGLLGLRIKYGRLFNRRRRRRRRR(SEQ ID NO: 68)  69 (S5)-R9 100 100 93EMFTILEEYFMYRGLLGLRIKYGRLFRRRRRRRRR (SEQ ID NO: 69)  70 (S6)-R9 100 10093 EMFTILEEYFMYRGLLGLRIKYGRLRRRRRRRRR (SEQ ID NO: 70)  71 (S7)-R9 91 8256 EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRRR (SEQ ID NO: 71)  72 (S8)-R9 100 10092 EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 72)  73 (S9)-R9 100 10084 EMFTILEEYFMYRGLLGLRIKYRRRRRRRRR (SEQ ID NO: 73)  74 (S10)-R9 100 10091 EMFTILEEYFMYRGLLGLRIKRRRRRRRRR  (SEQ ID NO: 74)  75 (S11)-R9 98 97 92EMFTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 75)  76 (S12)-R9 87 84 82EMFTILEEYFMYRGLLGLRRRRRRRRRR  (SEQ ID NO: 76)  77 (S13)-R9 93 89 84EMFTILEEYFMYRGLLGLRRRRRRRRR  (SEQ ID NO: 77)  79 (S14)-R9 97 93 85EMFTILEEYFMYRGLLRRRRRRRRR  (SEQ ID NO: 79)  80 (S15)-R9 91 87 78EMFTILEEYFMYRGLRRRRRRRRR  (SEQ ID NO: 80)  81 (S16)-R9 90 86 84EMFTILEEYFMYRGRRRRRRRRR  (SEQ ID NO: 81)  83 (S17)-R9 89 82 72EMFTILEEYFMYRRRRRRRRR  (SEQ ID NO: 83) 102 (S18)-R9 — 91 72MFTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 102) 103 (S19)-R9 — 99 93FTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 103) 104 (S20)-R9 — 93 74TILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 104) 105 (S21)-R9 — 91 62ILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 105) 106 (S22)-R9 — 92 70LEEYFMYGLLGLRIRRRRRRRRR  (SEQ ID NO: 106)

TABLE 3  Inhibition of TNF-αsecretion by S1-S22 peptides with an R⁹ sequence(SEQ ID NO: 379). RAW264.7 cells were plated at 3X10⁵ cells/well in 48-well plates.After 24 h the cells were incubated with peptide at various concentrations at roomtemperature in triplicate for 15 minutes and then stimulated with 1 ug/ml Cpg-ODN. Cells were then incubated for 4 hours at 37°C., supernatants collected, and TNF-αmeasured by ELISA. Percent inhibition was calculated by comparing TNF-αsecretionfrom cells incubated with peptide to control cells with no peptide treatment.Peptide 11 μM 7 μM 3.1 μM Sequence  42 (S1)-R9 83 67 37EMFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 42)  43 (S2)-R9 70 510 MFTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 43)  44 (S3)-R9 7824 0 FTILEEYFMYRGLLGLRIKYGRLFNEIRRRRRRRRR (SEQ ID NO: 44)  68 (S4)-R9 9583 69 EMFTILEEYFMYRGLLGLRIKYGRLFNRRRRRRRRR (SEQ ID NO: 68)  69 (S5)-R993 94 66 EMFTILEEYFMYRGLLGLRIKYGRLFRRRRRRRRR (SEQ ID NO: 69)  70 (S6)-R994 87 63 EMFTILEEYFMYRGLLGLRIKYGRLRRRRRRRRR (SEQ ID NO: 70)  71 (S7)-R938 29 21 EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRRR (SEQ ID NO: 71)  72 (S8)-R993 80 43 EMFTILEEYFMYRGLLGLRIKYGRRRRRRRRR (SEQ ID NO: 72)  73 (S9)-R9 8576 3 EMFTILEEYFMYRGLLGLRIKYRRRRRRRRR (SEQ ID NO: 73)  74 (S10)-R9 92 8035 EMFTILEEYFMYRGLLGLRIKRRRRRRRRR (SEQ ID NO: 74)  75 (S11)-R9 93 84 31EMFTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 75)  76 (S12)-R9 84 67 44EMFTILEEYFMYRGLLGLRRRRRRRRRR  (SEQ ID NO: 76)  77 (S13)-R9 81 77 18EMFTILEEYFMYRGLLGLRRRRRRRRR  (SEQ ID NO: 77)  79 (S14)-R9 86 77 0EMFTILEEYFMYRGLLRRRRRRRRR  (SEQ ID NO: 79)  80 (S15)-R9 81 68 57EMFTILEEYFMYRGLRRRRRRRRR  (SEQ ID NO: 80)  81 (S16)-R9 70 53 26EMFTILEEYFMYRGRRRRRRRRR  (SEQ ID NO: 81)  83 (S17)-R9 57 44 0EMFTILEEYFMYRRRRRRRRR  (SEQ ID NO: 83) 102 (S18)-R9 — 72 48MFTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 102) 103 (S19)-R9 — 3 87FTILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 103) 104 (S20)-R9 — 74 60TILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 104) 105 (S21)-R9 — 62 52ILEEYFMYRGLLGLRIRRRRRRRRR  (SEQ ID NO: 105) 106 (S22)-R9 70 53LEEYFMYGLLGLRIRRRRRRRRR  — (SEQ ID NO: 106)

Example 3 Effect of Peptides on of CpG-ODN-Induced Cytokine Secretion

Inhibition of TNF-α secretion by various T-peptides was examined.RAW264.7 cells were plated at 3×10⁵ cells/well in 48-well plates. After24 h the cells were incubated with peptide at various concentrations atroom temperature in triplicate for 15 minutes and then stimulated with1.25 ug/ml CpG-ODN or 1 ng/ml LPS. Cells were then incubated for 4 hoursat 37° C., supernatants collected, and TNF-α measured by ELISA. Percentinhibition was calculated by comparing TNF-α secretion from cellsincubated with peptide to control cells with no peptide treatment. Theinhibition by peptides T3, T11, T21, T36, T37, T51 and T52 were comparedto inhibition by an analogue of P13 having a sequence of nine arginineresidues (SEQ ID NO: 379) at the C-terminus (P13-R⁹; SEQ ID NO: 378).(Table 4).

TABLE 4  Percent Inhibition of TNF-α secretion by T peptides. Percentinhibition was calculated by comparing TNF-α secretion from cellsincubated with peptide to control cells with no peptide treatment.Peptide 22 μM 11.1 μM 7.1 μM Sequence CpG stimulation P13-R9 86 65 32DIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 378) T3-R9 99 92 85AIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 133) T21-R9 98 90 69DIVKLYVYDCIRRRRRRRRR (SEQ ID NO: 151) T36-R9 82 70 50DIIKLTVYDCIRRRRRRRRR (SEQ ID NO: 166) T37-R9 93 87 46DIVKVTVYDCIRRRRRRRRR (SEQ ID NO: 167) T51-R9 99.7 98 94AIVKLTVYACIRRRRRRRRR (SEQ ID NO: 181) T52-R9 100 99.2 97.8AIIKVYVYACIRRRRRRRRR (SEQ ID NO: 182) Peptide 22 μM 11.1 μM 7.4 μMSequence LPS Stimulation P13-R9 69 13 0 DIVKLTVYDCIRRRRRRRRR(SEQ ID NO: 378) T3-R9 77 65 33 AIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 133)T11-R9 91 81 68 DIVKLTVYACIRRRRRRRRR (SEQ ID NO: 141)

Example 4 Structure-Activity Testing of Peptides Derived from P13

Structure-activity relationships (SAR) were investigated to determinetolerances to residue modification in P13. Peptides used in the SARinclude SEQ ID NOS: 131-182, used to study the activities of SEQ ID NOS:317-368. Peptides possessing the following structural modifications weremade and evaluated for activity vis-à-vis P13:

1. Deletions of N- and/or C-terminal residues;

2. Alanine scan at each residue;

3. Comparison of residues with like charges at the same position (forexample, Asp or Glu at the same position; Lys, Arg, or H is at the sameposition);

4. Comparison of hydroxylated side chains at the same position (forexample, Ser or Thr at the same position);

5. Comparison of aromatic side chains at the same position (for example,Phe, Tyr, H is, or Trp at the same position);

6. Replacement of side chains with sulfur-containing side chains (forexample, Met and Cys);

7. Conservative replacement of branched aliphatic side chains (forexample, Leu, Ile, and Val);

8. Disruption of structure by inclusion of alternate residues;

9. Reverse and scrambled sequence; and

10. Dimerization.

The SAR revealed that certain substitutions led to peptides withactivity superior to that of P13. The substitution of a single aminoacid, in at least five different cases, and the substitution of two ormore amino acids in at least two cases, resulted in superior activity.These seven peptides are SEQ ID NOS: 133, 141, 151, 166, 167, 181, and182. The discovery was unexpected in that the activity of the SARderivatives was expected to be comparable or lesser than that of P13.Activity was defined as inhibition of TNF-α after stimulation ofRAW264.7 cells with CpG.

Example 5 Effect of Peptides on Otitis Media

Induction of Otitis Media:

BALB/c (Bagg albino) mice, about 8-12 weeks of age, are anesthetizedwith a subcutaneous injection of xylazine & ketamine (about 0.1 mg/30 gmbody weight) and their ears examined under the operating microscope toassure they are free of infection or perforation. One group of animalsis injected with PBS (Phospho-buffered saline) in one ear and with about10 μM of a peptide of Table 1 in the opposite ear, to determine theeffect of peptide without added bacteria. A second group of animalsreceive about 5.0 μl of PBS plus heat-inactivated S. pneumoniae (about10⁹ CFU/ml) in one ear and about 5.0 μl of peptide (about 10 μM) plusheat-inactivated S. pneumoniae (about 10⁹ CFU/ml) in the opposite ear.Injections are done through the tympanic membrane. Animals are killedabout 3 days after bacterial injection and tissue is histologicallyprocessed to assess middle ear disease. Inflammation is quantified bymeasuring 1) area of fluid present in the middle ear; 2) number of cellsin middle ear fluid; and 3) thickness of the tympanic membrane (TM)taken at a point away from the injection site. Data are obtained frommice (n=18) injected with PBS alone for each of the histologicalparameters measured, to serve as a control group. Disease induction isdefined as positive if the ear injected with S. pneumoniae withoutpeptide demonstrated an increase of at least two standard deviationsabove the control PBS treated mice in at least two of the threeparameters assessed for middle ear inflammation: Fluid area, cellnumber, thickness of the tympanic membrane.

Tissue Collection:

At the end of the experimental treatment, mice are killed and tissuescollected for histology. Mice are overdosed on anesthetic and perfusedintracardially with about 1.0 ml of saline, followed by about 20 ml offixative (about 1.5% paraformaldehyde, about 3% glutaraldehyde in about0.1 M phosphate buffer). The middle ears are left intact and connectedto each other by the skull base so both ears are processed together forhistology and sectioning. This enables all histologic embedding,sectioning, staining, and analysis to be done on the two sidessimultaneously to reduce any impact of processing variables on thesubsequent quantitative analyses. Middle ears are decalcified, embeddedin glycol methacrylate plastic, sectioned at about 5 μm, mountedserially on glass slides, stained, and coverslipped.

Histopathologic Analysis:

Three consecutive sections at the level of the umbo and promontory areselected for measures of 1) area of fluid present in the middle ear; 2)number of cells in middle ear fluid; and 3) thickness of the tympanicmembrane. Each measurement is taken on the three sequential sections perspecimen.

Statistical Analyses:

To determine the effect of peptide without added bacteria, animals areinjected in one ear with PBS alone, and the other ear with about 10 μMpeptide. Paired t-tests are done comparing the effect of PBS alone withthe effect of peptide for each of the three histological parameters: 1)area of fluid present in the middle ear; 2) number of cells in middleear fluid; and 3) thickness of the tympanic membrane. Paired t-tests aredone using these animals comparing the effect of peptide plus S.pneumoniae in one ear with S. pneumoniae alone in the opposite ear foreach of the histological parameters described above.

Example 6 Effect of Peptides on Septic Shock

Inhibition of Inflammatory Mediators in a Murine Septic Shock Model.

BALB/c mice are injected i.p. with PBS, LPS at about 100 μg/mouse/250μl, or about 100 μg LPS plus various doses of peptides. Serum iscollected at about 2 and about 6 hours after treatment and evaluated forthe pro-inflammatory cytokines MIP-2 and TNF-α by ELISA, and for solubleICAM-1.

Example 7 Effects of P13 on Mouse Middle Ear

Assay of Effects Caused by Peptide without Added Bacteria.

Five mice were injected in one ear with PBS and in the opposite ear with10 μM peptide P13. Three days later the animals were killed, and themiddle ears embedded, sectioned, stained and evaluated for fluid area,infiltrating cell number, and thickness of the tympanic membrane. Pairedt-tests (2-tailed) were used to analyze each of the three parameters. Inthe absence of bacterial-induced inflammation, no differences were seenbetween the PBS-injected ear and P13-injected ear in 1) fluid area(p=0.104); 2) cell number (p=0.880); or 3) tympanic membrane thickness(p=0.891).

Assay of Effects Caused by Peptide with Added Bacteria.

To examine the effectiveness of the peptide to affect inflammation invivo, twenty BALB/c mice were injected in the middle ear on one sidewith heat-inactivated S. pneumoniae plus PBS, and in the middle ear onthe opposite side with heat-inactivated S. pneumoniae plus 10 μM peptideP13. Three days later the animals were killed, and evaluated for middleear fluid area, infiltrating cell number, and thickness of the tympanicmembrane. Disease development was defined as an increase over backgroundcontrols (PBS injected ears n=18) of at least two standard deviations intwo out of the three parameters quantified. A total of 7 out of 20 micemet the criteria for disease induction. Analysis of middle ears bypaired t-tests from these 7 mice with disease showed that peptidetreatment significantly reduced the amount of fluid (p=0.004),infiltrating cell number (p=0.02), and thickness of the tympanicmembrane (p=0.002). Examination of these three parameters ofinflammation for each individual mouse with disease illustrated thedramatic effect seen with a single treatment of peptide P13. Ofinterest, 6 of the 7 mice demonstrated reductions in all areas ofinflammation, while one animal showed only modest reduction in fluidarea and tympanic membrane thickness, and no reduction in cell number.Injection of heat-killed bacteria resulted in a marked inflammatoryresponse in the middle ear after 3 days. This was characterized bymucosal and tympanic membrane swelling, cellular infiltration, andsignificant fluid (effusion) secretion and accumulation that filled themiddle ear space. The inflammatory response led to significant mucosalcellular hypertrophy and active secretion of mucins and other fluids.When peptide P13 was injected with the bacteria, a significant reductionwas seen in fluid accumulation into the middle ear space and reducedmucosal hypertrophy.

TABLE 5 Peptide Inhibition of Middle Ear Inflammation^(a) Cell TympanicMembrane Fluid Area Number Thickness (microns ± Treatment (microns² ±S.D.) (±S.D.) S.D.) PBS^(b) 1016 ± 1397 31 ± 41 44 ± 20 S. pneumoniae^(c) 5771 ± 2077 252 ± 140 105 ± 33  S. pneumoniae + 1486 ± 1192 111 ±119 44 ± 15 peptide^(c) p value (2-tailed)^(d) p = 0.004 p = 0.020 p =0.002 ^(a)Middle ear inflammation is assessed by measuring threeconsecutive tissue sections for area of fluid in the middle ear, numberof cells in the middle ear fluid, and thickness of the tympanic membranemeasured at a point away from the injection site. Data represent themean ± SD animals with middle ear inflammation. Statistical evaluationis done using a paired t-test. ^(b)The PBS treated animals receive nobacteria or peptides. ^(c)Animals are injected in one ear with S.pneumoniae + PBS and in the opposite ear with S. pneumoniae + peptide(about 10 μM). ^(d)Statistical evaluation using a paired t-test is doneusing data collected from diseased animals injected with bacteria andcomparing peptide vs. no peptide treatment.

Example 8 Assay for Crossing the Tympanic Membrane by FITC-LabeledP13-R⁹ (SEQ ID NO: 378)

BALB/c mice were injected through the bulla by the following protocol.

Three BALB/c mice at 8-12 weeks of age were anesthetized with ketamine(100 mg/kg) and xylazine (20 mg/kg) prior to administration of bacteria.A ventral midline incision was made in the neck, and the bulla exposedafter blunt dissection. The middle ear was inoculated through the bonywall with approximately 3.5 μl of a bacterial suspension (heatinactivated S. pneumoniae 10¹⁰ CFU/ml) with a thin needle. Both theright and left ears received bacteria by bulla injection.

After 24 hours, animals received an otoscopic exam using an operatingmicroscope. Mice were anesthetized as described above, and the tympanicmembrane was inspected for the presence or absence of the followingthree criteria for acute otitis media (AOM): 1) middle ear effusionbehind the tympanic membrane; 2) change in color of the tympanicmembrane from clear to red or white (indicating inflammation and/orpurulence in the middle ear); 3) change in position of the tympanicmembrane from neutral to bulging or retracted. Only animalsdemonstrating AOM by these criteria were used.

Four ears met the criteria for AOM. FITC labeled peptide P13-R⁹ (SEQ IDNO: 378) (10 μg/30 μl) was dropped onto the middle ear. The peptide wasallowed to absorb for 10 minutes. After 10 minutes, the external earcanal was flushed twice, and blotted to get rid of any remainingpeptide. The middle ear fluid was then aspirated with a 10 μl Hamiltonsyringe with a 30 g needle, and the fluid smeared on a slide.FITC-labeled peptide was viewed under a fluorescent scope. Thephotograph demonstrates that FITC-labeled P13-R⁹ (SEQ ID NO: 378)crosses the tympanic membrane and associates with cells in middle earfluid.

FIG. 1 illustrates bright field microscopy of cells in middle ear fluid(Panel A). FIG. 1 illustrates fluorescent microscopy of FITC-labeledP13-R⁹ (SEQ ID NO: 378) associated with cells in middle ear fluid (PanelB).

Example 9 Topical Administration of P13 Reduced Middle Ear Inflammationand Fluid Retention

In this experiment, P13 was administered by ear drops to reduceinflammation and fluid retention in the pre-clinical AOM model. BALB/cmice (n=13) received an otoscopic exam using an operating microscope toestablish the clinical symptoms of AOM. Mice were anesthetized and thetympanic membrane (TM) inspected for presence or absence of thefollowing 3 criteria for AOM: 1) middle ear effusion behind the TM; 2)change in color of the TM from clear to red or white, indicatinginflammation and/or purulence in the middle ear; and 3) change inposition of the TM from neutral to bulging or retracted. An animalexhibiting any of these changes was scored positive for inflammation.All mice scored negative on pre-screen and were injectedtranstympanically with 5 μl heat inactivated S. pneumonia (10⁹ CFU/ml)in both ears. Twenty-four hours post bacterial injection, mice wereexamined by otoscopic exam and those animals meeting the inflammationcriteria described above remained in the study. Of the thirteen earsscored as positive; seven ears were treated topically with P13 (1 μg/30μl), and six ears treated topically with PBS (30 μl). For topicaltreatment, animals were lightly anesthetized and P13 or PBS was droppedonto the external tympanic membrane. The animals remained sedated for15-20 minutes. Seventy-two hours post bacterial injection, animals weresacrificed and tissue histologically processed to assess middle eardisease. Inflammation was quantified by measuring the area of fluidpresent in the middle ear and number of cells in the measured middle earfluid area as follows. Mice were overdosed on anesthetic and perfusedintracardially with 1.0 ml of saline, followed by 20 ml of fixative(1.5% paraformaldehyde-3% glutaraldehyde in 0.1 M phosphate buffer).Middle ears were decalcified, embedded in glycol methacrylate plastic,sectioned at 5 μm, mounted serially on glass slides stained, andcover-slipped. Three consecutive sections at the level of the umbo andpromontory were selected for measures of 1) area of fluid present in themiddle ear; and 2) number of cells in middle ear fluid. Each measurementwas taken on the three sequential sections per specimen. The valuepresented for each parameter represents the mean of the three sections(FIG. 2). This experiment confirmed that ear drop administration of P13dramatically reduced both the cellular infiltration and residual fluidin the pre-clinical AOM model. Of interest, the one animal in the P13treatment group showing high cell number and high fluid area is the sameanimal.

Example 10 Topical Administration of P13 Significantly Reduced theSeverity and Longevity of Hearing Impairment

A. Topical Administration of P13 Demonstrated Efficacy in a Mouse Modelof AOM.

Patients with AOM frequently experience residual middle ear fluidretention which can lead to impaired hearing, recurrent infections, andin extreme conditions the need for surgical placement of ear tubes. Todetermine whether topical administration of P13 would impact hearing ina pre-clinical model of AOM, BALB/c mice (n=8), 13 weeks of age,received a baseline auditory brainstem response (ABR) and otoscopic examin both ears. ABR stimuli consisted of 20 tone-burst trains at 4 kHz, 8kHz, 16 kHz and 32 kHz at five intensity levels in 10 dB steps. Eachtone-burst had a two-ms duration, with tone burst onsets separated by 12ms. Two separate trains offset by 5 dB were presented as stimuli, thencombined in data analysis to determine threshold in 5 dB steps.Responses to 300 stimulus repetitions were averaged using a digitaloscilloscope. Thresholds were based on the lowest intensity at which aresponse could be identified. The number of waves present at thresholdvaried somewhat, but the presence of at least two waves was considered avalid threshold. An otoscopic exam was performed using an operatingmicroscope to establish the clinical symptoms of AOM, as previouslydescribed. All 8 animals remained in the study and were injectedtranstympanically with 5 μl heat inactivated S. pneumonia (10⁹ CFU/ml)in both ears. Twenty-four hours post bacterial injection, mice wereexamined by otoscopic exam and those animals meeting the inflammationcriteria remained in this study. After 24 hours, each animal was treatedtopically with P13 (1 μg/30 μl) in one ear, and PBS (30 ul) in theother. For topical treatment, animals were lightly anesthetized and P13or PBS dropped onto the external tympanic membrane. The animals remainedsedated for 15-20 minutes.

Five and 13 days after bacterial injection, all animals received ABRtesting as described above. ABR data was calculated by subtracting thebaseline ABR from the post-bacterial ABR for each frequency, and thensumming all 4 frequencies (4, 8, 16 and 32 kHz). A two-way repeatedmeasure ANOVA was done for data analysis using days 5 and 13. Thisexperiment confirmed that ear drop administration of P13 significantlylimited the hearing impairment seen in control treated animals.Treatment with P13 reduced the severity of hearing impairment andreduced the time to resolution to normal baseline hearing levels (FIG.3A). P13 treatment dramatically reduced the number of animals withhearing loss as compared to PBS treatment (FIG. 3B). In summary, thesedata confirmed the efficacy of P13 in reducing the severity andlongevity of hearing impairment.

B. Topical Administration of P13 Demonstrated Efficacy in a Mouse Modelof Chronic Otitis Media.

Eight C3H/HeJ mice (12 months of age) were given a clinical earexamination. Those animals demonstrating middle ear inflammation (6mice) were given two baseline ABRs, one week apart, and then each earwas treated topically with either PBS, or 1 μg P13. One and two weeksafter P13/PBS treatment, animals again received an otoscopic exam and anABR assessment. ABR data were calculated by subtracting the baseline ABRfrom the post-treatment ABR for each frequency, and then summing all 4frequencies. Topical P13 treatment resulted in a statisticallysignificant improvement in hearing thresholds as assessed by ABRmeasurements across all four frequencies at both weeks 1 and 2post-treatment. This data showed a dramatic improvement in hearingthresholds at both time-points, with an approximate 40 db improvement atweek 2. An examination of a single frequency (4 kHz) demonstrated theimpact of P13 treatment on hearing, where at week 2 post-treatment,there was an approximate 12 db hearing improvement in P13 treatedanimals as compared to controls. No change was observed in the middleear inflammatory status as assessed by otoscopic exam after peptidetreatment at either time point.

C. Comparison of the D- and L-Isomer Forms of P13 in Treating COM

The biological half-life of peptides can be improved by using theD-isomer of amino acid residues in place of the L-isomer form. Astereoisomer of P13 containing all D-amino acid residues (D-P13) wastherefore tested for efficacy in treating C3H/HeJ mice with COM, and theresults were compared to those obtained with the stereoisomer containingall L-amino acid residues (L-P13). The D-P13 peptide was produced bystandard methods and was purified to >95%. Three C3H/HeJ mice with COMwere treated topically with D-P13 in both ears as described above andABRs assessed at weeks 1 and 2 post-treatment. Similar to what was seenwith L-P13 treatment, treatment with D-P13 also demonstrated efficacy inimproving hearing thresholds in COM mice with clinically documenteddisease. At week 2 post-treatment, D-P13 improved hearing thresholdsacross all frequencies approximately 20 db as compared to an approximate40 db improvement after treatment with L-P13. While the L-isomer of P13showed a more pronounced improvement of hearing thresholds, the D- andL-isomer forms of P13 exhibited similar levels of statistical confidencein this experiment.

Example 11 Topical Administration of P13 Lacked Ototoxicity

P13 was investigated for possible negative impact on hearing in normalmice upon administration by ear drops. BALB/c mice, 16 weeks of age,(n=16) received two baseline auditory brainstem response (ABR) tests oneweek apart, and an otoscopic exam in both ears. ABR stimuli consisted of20 tone-burst trains at 4 kHz, 8 kHz, 16 kHz and 32 kHz at fiveintensity levels in 10 dB steps, as previously described. An otoscopicexam was performed using an operating microscope to establish theclinical symptoms of AOM, as previously described. Any animal with anabnormal ABR or exam on pre-screen was not entered into the study.

All 16 animals remained in the study and were divided into five groupsas follows:

group 1: (n=6 ears) 1 μg P13/30 μl, topically, given once;

group 2: (n=7 ears) 10 μg P13/30 μl, topically, given once;

group 3: (n=7 ears) 10 μg P13/30 μl, topically, given twice, 24 hoursapart;

group 4: (n=6 ears) 60 μg P13/30 μl, topically, given once; and

group 5: (n=6 ears) untreated mice.

ABRs were performed at one, two, three and four weeks post P13treatment. The two baseline ABRs were averaged and compared to the ABRspost P13 treatment. This experiment demonstrated that topicaladministration of P13 did not negatively impact hearing thresholds inthese normal mice.

Example 12 Evaluation of TNF-α Secretion in Mice Treated with Peptides

The effect of several peptides on TNF-α secretion in mice in response tostimulation by CpG-ODN and LPS is studied. Peptide compositions areprepared for each peptide-R⁹ (SEQ ID NO: 379), at 3 concentrations, 37μM, 22.2 μM, and 11.1 μM, and tested. Peptides demonstrating activity inthis assay are further formulated into peptide compositions at 11.1 μM,7.4 μM, and 3.7 μM for re-assay.

BALB/c (Bagg albino) mice, about 8-12 weeks of age, are anesthetizedwith a subcutaneous injection of xylazine & ketamine (about 0.1 mg/30 gmbody weight). The mice are injected in both ears with either 1.25 ug/mlCpG-ODN or 1 ng/ml LPS. Twenty-four hours later the mice are treatedtopically (ear drop) with 1 μg peptide/30 μl in one ear and PBS (30 μl)in the other. Twenty-four hours post peptide/PBS treatment, middle earfluid is collected and analyzed for TNF-α secretion by ELISA. Earsexhibiting lesser TNF-α secretion than the control ears identifypeptides that effectively inhibit TNF-α secretion in the live mouse.

EMBODIMENTS

The following embodiments provide non-limiting examples of objects ofthe invention.

In some embodiments, the invention contemplates a pharmaceuticalcomposition comprising a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue.

In some embodiments, the pharmaceutical composition of claim 1, whereinthe peptide further comprises a transducing sequence at the C-terminus.

In some embodiments, the transducing sequence is a poly-argininesequence.

In some embodiments, the transducing sequence comprises nine consecutivearginine residues (SEQ ID NO: 379).

In some embodiments, the transducing sequence consists of nineconsecutive arginine residues (SEQ ID NO: 379).

In some embodiments, the peptide comprises a derivative of P13comprising at least one D-amino acid residue.

In some embodiments, the amino acid residues of the peptide are D-aminoacid residues.

In some embodiments, the amino acid residues that are not part of thetransducing sequence are D-amino acid residues.

In some embodiments, at least one amino acid residue of the transducingsequence is a D-amino acid residue.

In some embodiments, all the amino acid residues of the transducingsequence are D-amino acid residues.

In some embodiments, the peptide is any one of SEQ ID NOS: 42-44, 68-77,79-81, 83, 102-106, 133, 141, 151, 166, 167, 181, and 182.

In some embodiments, the peptide is any one of SEQ ID NOS: 228-230,254-263, 265-267, 269, 288-292, 319, 327, 337, 352, 353, 367, and 368.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition is in the form of adrop.

In some embodiments, the pharmaceutical composition is in the form of anaerosol, vapor, spray, or mist.

In some embodiments, the invention contemplates a pharmaceuticalcomposition comprising a peptide comprising a sequence of any one of SEQID NOS: 1-186.

In some embodiments, the invention contemplates a peptide comprising thesequence of any one of SEQ ID NOS: 1-368.

In some embodiments, the peptide comprises the sequence of any one ofSEQ ID NOS: 42-44, 68-77, 79-81, 83, 102-106, 133, 141, 151, 166, 167,181, 182, 228-230, 254-263, 265-267, 269, 288-292, 319, 327, 337, 352,353, 367, and 368.

In some embodiments, the invention contemplates a derivative of apeptide comprising the sequence of any one of SEQ ID NOS: 1-369, whereinthe derivative comprises at least one D-amino acid residue.

In some embodiments, the invention contemplates a method of regulatingcellular activity, the method comprising administering to an organism inneed or want thereof an effective amount of a pharmaceutical compositioncomprising a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the cellular activity is mediated by a toll-likereceptor.

In some embodiments, the cellular activity mediated by a toll-likereceptor is TNF-α secretion.

In some embodiments, the method provides 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90% inhibition of TNF-α secretion following stimulation byLPS and/or CpG-ODN.

In some embodiments, the administering results in an inhibition ofcytokine secretion.

In some embodiments, the invention contemplates a method of treatinginflammation in an animal, the method comprising administering to ananimal in need or want thereof a pharmaceutical composition comprising apeptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the inflammation is caused by a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof.

In some embodiments, the inflammation is ear inflammation.

In some embodiments, the inflammation is otitis media.

In some embodiments, the administration reduces or eliminates a symptomof otitis media.

In some embodiments, the symptom is pain, otorrhea, fever, irritability,anorexia, vomiting or diarrhea.

In some embodiments, the symptom is pain.

In some embodiments, the inflammation is inflammation of the skin,joints, muscular tissue, brain, or connective tissue.

In some embodiments, the inflammation is arthritis, dermatitis, Lupuserythematosus, meningitis, or psoriasis.

In some embodiments, the arthritis is osteoarthritis, rheumatoidarthritis, septic arthritis, gout, pseudo-gout, juvenile idiopathicarthritis, Still's disease, or ankylosing spondylitis.

In some embodiments, the dermatitis is spongiotic dermatitis, childhoodeczema, allergic contact dermatitis, seborrhoeic dermatitis, dyshidroticdermatitis, urticaria, vesicular or bullous dermatitis, or papularurticaria.

In some embodiments, the psoriasis is plaque psoriasis, flexuralpsoriasis, guttate psoriasis, pustular psoriasis, nail psoriasis,psoriatic arthritis, or erythrodermic psoriasis.

In some embodiments, the pharmaceutical composition is administered viatopical application.

In some embodiments, the topical application comprises application tothe skin, hair, outer ear, tympanic membrane, buccal cavity, nasalcavity, or sublingual cavity.

In some embodiments, the topical application comprises application tothe tympanic membrane.

In some embodiments, the application to the tympanic membrane comprisesthe application of drops to the tympanic membrane.

In some embodiments, the animal is a human.

In some embodiments, the invention contemplates a method of treatingsinusitis, the method comprising administering an aerosol composition toan organism in need or want thereof, the aerosol composition comprisinga therapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a method of improvinghearing in an animal, the method comprising administering to an animalhaving middle and/or inner ear inflammation and reduced hearing atherapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus, wherein the peptide is administered topically,wherein the hearing improves to a level no better than ordinary levels,and/or the hearing improves faster than the hearing would improvewithout administration of the peptide.

In some embodiments, the invention contemplates a method of treatingmiddle and/or inner ear inflammation, the method comprisingadministering to a tympanic membrane of an animal in need or wantthereof a therapeutically-effective amount of a peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for regulating cellular activity, thepeptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the cellular activity is mediated by a toll-likereceptor.

In some embodiments, the cellular activity mediated by a toll-likereceptor is TNF-α secretion.

In some embodiments, the medicament provides 10%, 20%, 30%, 40%, 50%,60%, 70%, 80% or 90% inhibition of TNF-α secretion following stimulationby LPS and/or CpG-ODN.

In some embodiments, the medicament is suitable to inhibit cytokinesecretion.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating inflammation in an animal,the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-368; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the inflammation is caused by a virus, bacteria,fungi, antigen, self-antigen, or a combination thereof.

In some embodiments, the inflammation is ear inflammation.

In some embodiments, the inflammation is otitis media.

In some embodiments, the administration reduces or eliminates a symptomof otitis media.

In some embodiments, the symptom is pain, otorrhea, fever, irritability,anorexia, vomiting or diarrhea.

In some embodiments, the symptom is pain.

In some embodiments, the inflammation is inflammation of the skin,joints, muscular tissue, brain, or connective tissue.

In some embodiments, the inflammation is arthritis, dermatitis, Lupuserythematosus, meningitis, or psoriasis.

In some embodiments, the arthritis is osteoarthritis, rheumatoidarthritis, septic arthritis, gout, pseudo-gout, juvenile idiopathicarthritis, Still's disease, or ankylosing spondylitis.

In some embodiments, the dermatitis is spongiotic dermatitis, childhoodeczema, allergic contact dermatitis, seborrhoeic dermatitis, dyshidroticdermatitis, urticaria, vesicular or bullous dermatitis, or papularurticaria.

In some embodiments, the psoriasis is plaque psoriasis, flexuralpsoriasis, guttate psoriasis, pustular psoriasis, nail psoriasis,psoriatic arthritis, or erythrodermic psoriasis.

In some embodiments, the medicament is suitable for topical application.

In some embodiments, the topical application comprises application tothe skin, hair, outer ear, tympanic membrane, buccal cavity, nasalcavity, or sublingual cavity.

In some embodiments, the topical application comprises application tothe tympanic membrane.

In some embodiments, the application to the tympanic membrane comprisesthe application of drops to the tympanic membrane.

In some embodiments, the animal is a human.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating sinusitis, the medicamentcomprising an aerosol composition, the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for improving hearing in an animal,wherein the animal has middle and/or inner ear inflammation and reducedhearing, the peptide comprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus, wherein the medicament is a topical medicament.

In some embodiments, the invention contemplates a use of a peptide inthe manufacture of a medicament for treating middle and/or inner earinflammation, wherein the medicament is suitable for administration to atympanic membrane of an animal in need or want thereof, the peptidecomprising:

a) a sequence of any one of SEQ ID NOS: 187-369; or

b) a derivative of P13 comprising at least one D-amino acid residue,

wherein the peptide optionally further comprises a transducing sequenceat the C-terminus.

Embodiment 101

A composition comprising a peptide comprising the amino acid sequenceFTILEEYFMY (SEQ ID NO: 371).

Embodiment 102

The composition of embodiment 101, wherein the peptide is selected fromthe group consisting of S1-S17.

Embodiment 103

The composition of embodiment 101, wherein peptide further comprises atransducing sequence.

Embodiment 104

The composition of embodiment 103, wherein the transducing sequencecomprises a 9-arginine sequence positioned at the C-terminus.

Embodiment 105

A method of regulating cellular activity comprising administering apeptide to a cell, said peptide comprising the amino acid sequenceFTILEEYFMY (SEQ ID NO: 371).

Embodiment 106

The method of embodiment 105, wherein the peptide is selected from thegroup consisting of S1-S17.

Embodiment 107

The method of embodiment 105, wherein the peptide further comprises atransducing sequence.

Embodiment 108

The method of embodiment 106, wherein the transducing sequence comprisesa 9-arginine sequence positioned at the C-terminus.

Embodiment 109

The method of embodiment 105, wherein the activity is mediated bytoll-like receptor.

Embodiment 110

The method of embodiment 105, wherein the administration results in aninhibition of cytokine secretion.

Embodiment 111

The method of embodiment 106, wherein the peptide is used in thetreatment of inflammation.

Embodiment 112

The method of embodiment 111, wherein the inflammation is caused by avirus, bacteria, fungi, antigen, self-antigen, or a combination thereof.

Embodiment 113

The method of embodiment 105, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by CpG-ODN.

Embodiment 114

A method of decreasing TNF-α secretion in response to toll-like receptorsignaling comprising administering a therapeutically effective dose of apeptide derived from A52R.

Embodiment 115

A pharmaceutical composition comprising:

a peptide comprising the amino acid sequence set forth in any one of thepeptides selected from S1-S17;

a 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus ofthe said peptide; and

a pharmaceutically-acceptable excipient,

wherein said composition is used for the treatment inflammation.

Embodiment 201

A composition comprising a peptide comprising the amino acid sequenceLEEYFMY (SEQ ID NO: 370).

Embodiment 202

The composition of embodiment 201, wherein the peptide is selected fromthe group consisting of S1-S22.

Embodiment 203

The composition of embodiments 201 or 202, wherein peptide furthercomprises a transducing sequence.

Embodiment 204

The composition of embodiment 203, wherein the transducing sequencecomprises a 9-arginine sequence (SEQ ID NO: 379) positioned at theC-terminus.

Embodiment 205

The composition of embodiments 201 or 202, wherein the peptide comprisesa L-isomer amino acid or a D-isomer amino acid or L- and D-isomer aminoacids.

Embodiment 206

The composition of embodiment 203, wherein the peptide and/or thetransducing sequence comprises a L-isomer amino acid or a D-isomer aminoacid or L- and D-isomer amino acids.

Embodiment 207

A method of regulating cellular activity comprising administering apeptide to a cell, said peptide comprising the amino acid sequenceLEEYFMY (SEQ ID NO: 370).

Embodiment 208

The method of embodiment 207, wherein the peptide is selected from thegroup consisting of S1-S22.

Embodiment 209

The method of embodiments 207 or 208, wherein the peptide furthercomprises a transducing sequence.

Embodiment 210

The method of embodiment 209, wherein the transducing sequence comprisesa 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus.

Embodiment 211

The method of embodiments 207 or 208, wherein the peptide comprises aL-isomer amino acid or a D-isomer amino acid or L- and D-isomer aminoacids.

Embodiment 212

The method of embodiment 209, wherein the peptide and/or the transducingsequence comprises a L-isomer amino acid or a D-isomer amino acid or L-and D-isomer amino acids.

Embodiment 213

The method of embodiment 207, wherein the activity is mediated bytoll-like receptor.

Embodiment 214

The method of embodiment 207, wherein the administration results in aninhibition of cytokine secretion.

Embodiment 215

The method of embodiment 208, wherein the peptide is used in thetreatment of inflammation.

Embodiment 216

The method of embodiment 215, wherein the inflammation is caused by avirus, bacteria, fungi, antigen, self-antigen, or a combination thereof.

Embodiment 217

The method of embodiment 207, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by CpG-ODN.

Embodiment 218

A method of decreasing TNF-α secretion in response to toll-like receptorsignaling comprising administering a therapeutically effective dose of apeptide derived from A52R.

Embodiment 219

A pharmaceutical composition comprising: (a) a peptide comprising theamino acid sequence set forth in any one of the peptides selected fromS1-S22; (b) a 9-arginine sequence (SEQ ID NO: 379) positioned at theC-terminus of the said peptide; and a pharmaceutically-acceptableexcipient, wherein said composition is used for the treatmentinflammation.

Embodiment 301

A composition comprising a peptide comprising the amino acid sequenceVYDCI (SEQ ID NO: 372), VYACI (SEQ ID NO: 373), KLTVY (SEQ ID NO: 374),KLYVY (SEQ ID NO: 375), or KVYVY (SEQ ID NO: 376).

Embodiment 302

The composition of embodiment 301, wherein the peptide is selected fromthe group consisting of the peptides presented in Table 1.

Embodiment 303

The composition of embodiment 301, wherein peptide further comprises atransducing sequence.

Embodiment 304

The composition of embodiment 303, wherein the transducing sequencecomprises a 9-arginine sequence (SEQ ID NO: 379) positioned at theC-terminus.

Embodiment 305

A method of regulating cellular activity comprising administering apeptide to a cell, said peptide comprising the amino acid sequence VYDCI(SEQ ID NO: 372), VYACI (SEQ ID NO: 373), KLTVY (SEQ ID NO: 374), KLYVY(SEQ ID NO: 375), or KVYVY (SEQ ID NO: 376).

Embodiment 306

The method of embodiment 305, wherein the peptide is selected from thegroup consisting of the peptides presented in Table 1.

Embodiment 307

The method of embodiment 305, wherein the peptide further comprises atransducing sequence.

Embodiment 308

The method of embodiment 306, wherein the transducing sequence comprisesa 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus.

Embodiment 309

The method of embodiment 305, wherein the activity is mediated bytoll-like receptor.

Embodiment 310

The method of embodiment 305, wherein the administration results in aninhibition of cytokine secretion.

Embodiment 311

The method of embodiment 306, wherein the peptide is used in thetreatment of inflammation.

Embodiment 312

The method of embodiment 311, wherein the inflammation is caused by avirus, bacteria, fungi, antigen, self-antigen, or a combination thereof.

Embodiment 313

The method of embodiment 305, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by CpG-ODN.

Embodiment 314

The method of embodiment 305, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by LPS.

Embodiment 315

A method of decreasing TNF-α secretion in response to toll-like receptorsignaling comprising administering a therapeutically effective dose of apeptide derived from P13.

Embodiment 316

A pharmaceutical composition comprising:

a peptide comprising the amino acid sequence set forth in any one of thepeptides selected from the peptides listed in Table 1;

a 9-arginine sequence positioned at the C-terminus of the said peptide;and

a pharmaceutically-acceptable excipient,

wherein said composition is used for the treatment inflammation.

Embodiment 401

A composition comprising a peptide comprising the amino acid sequenceVYACI (SEQ ID NO: 373), KLYVY (SEQ ID NO: 375), or KVYVY (SEQ ID NO:376).

Embodiment 402

The composition of embodiment 401, wherein the peptide is selected fromthe group consisting of the peptides presented in Table 1.

Embodiment 403

The composition of embodiments 401 or 402, wherein the peptide furthercomprises a transducing sequence.

Embodiment 404

The composition of embodiment 403, wherein the transducing sequencecomprises a 9-arginine sequence (SEQ ID NO: 379) positioned at theC-terminus.

Embodiment 405

The composition of embodiments 401 or 402, wherein the peptide comprisesa L-isomer amino acid or a D-isomer amino acid or L- and D-isomer aminoacids.

Embodiment 406

The composition of embodiment 403, wherein the peptide and/or thetransducing sequence comprises a L-isomer amino acid or a D-isomer aminoacid or L- and D-isomer amino acids.

Embodiment 407

A method of regulating cellular activity comprising administering apeptide to a cell, said peptide comprising the amino acid sequence VYACI(SEQ ID NO: 373), KLYVY (SEQ ID NO: 375), or KVYVY (SEQ ID NO: 376).

Embodiment 408

The method of embodiment 407, wherein the peptide is selected from thegroup consisting of the peptides presented in Table 1.

Embodiment 409

The method of embodiments 407 or 408, wherein the peptide furthercomprises a transducing sequence.

Embodiment 410

The method of embodiment 409, wherein the transducing sequence comprisesa 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus.

Embodiment 411

The method of embodiments 407 or 408, wherein the peptide comprises aL-isomer amino acid or a D-isomer amino acid or L- and D-isomer aminoacids.

Embodiment 412

The method of embodiment 409, wherein the peptide and/or the transducingsequence comprises a L-isomer amino acid or a D-isomer amino acid or L-and D-isomer amino acids.

Embodiment 413

The method of embodiment 407, wherein the activity is mediated bytoll-like receptor.

Embodiment 414

The method of embodiment 407, wherein the administration results in aninhibition of cytokine secretion.

Embodiment 415

The method of embodiment 408, wherein the peptide is used in thetreatment of inflammation.

Embodiment 416

The method of embodiment 415, wherein the inflammation is caused by avirus, bacteria, fungi, antigen, self-antigen, or a combination thereof.

Embodiment 417

The method of embodiment 407, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by CpG-ODN.

Embodiment 418

The method of embodiment 407, wherein the administration of said peptideto said cell results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%inhibition of TNF-α secretion following stimulation by LPS.

Embodiment 419

A method of decreasing TNF-α secretion in response to toll-like receptorsignaling comprising administering a therapeutically effective dose of apeptide derived from P13.

Embodiment 420

A pharmaceutical composition comprising:

a peptide comprising the amino acid sequence set forth in any one of thepeptides selected from the peptides listed in Table 1;

a 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus ofthe said peptide; and

a pharmaceutically-acceptable excipient,

wherein said composition is used for the treatment inflammation.

Embodiment 501

A pharmaceutical composition comprising:

a peptide comprising the amino acid sequence set forth in any one of thepeptides selected from P13, a P13 variant, derivative, stereoisomer, oranalogue, or a peptide of Table 1;

a 9-arginine sequence (SEQ ID NO: 379) positioned at the C-terminus ofthe said peptide; and

a pharmaceutically-acceptable excipient,

wherein said composition is used for the treatment of inflammation.

Embodiment 502

The pharmaceutical composition of embodiment 501, wherein theinflammation comprises otitis media.

Embodiment 503

The pharmaceutical composition of embodiment 502, further comprisingwherein the pharmaceutical composition is applied via topicalapplication.

Embodiment 504

The pharmaceutical composition of embodiment 503, wherein the topicalapplication comprises application to the skin, hair, outer ear, tympanicmembrane, buccal cavity, or sublingual cavity.

Embodiment 505

The pharmaceutical composition of embodiment 504, wherein the topicalapplication comprises application to the tympanic membrane.

Embodiment 506

The pharmaceutical composition of embodiment 505, wherein theapplication to the tympanic membrane comprises the application of eardrops to the tympanic membrane.

Embodiment 507

A pharmaceutical composition effective to:

decrease the amount of middle ear fluid by about 30-80 percent;

decrease an infiltrating cell number in middle ear fluid by about 30-80percent; and

decrease the thickness of a tympanic membrane by about 30-80 percent ina mouse comprising inflammation of the ear, wherein the composition isadministered to the mouse in an amount of about 0.01 ug to 60 ug.

Embodiment 508

The pharmaceutical composition of embodiment 507, comprising a peptide.

Embodiment 509

The pharmaceutical composition of embodiment 508, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 510

The pharmaceutical composition of embodiment 509, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 511

The pharmaceutical composition of embodiment 509, wherein the peptidecomprises P13.

Embodiment 512

The pharmaceutical composition of embodiment 509, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 513

The pharmaceutical composition of embodiment 512, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 514

The pharmaceutical composition of embodiment 507, wherein theinflammation comprises otitis media.

Embodiment 515

The pharmaceutical composition of embodiment 514, comprising a peptide.

Embodiment 516

The pharmaceutical composition of embodiment 515, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 517

The pharmaceutical composition of embodiment 516, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 518

The pharmaceutical composition of embodiment 516, wherein the peptidecomprises P13.

Embodiment 519

The pharmaceutical composition of embodiment 516, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 520

The pharmaceutical composition of embodiment 519, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 521

The pharmaceutical composition of embodiment 514, further comprisingwherein the pharmaceutical composition is applied via topicalapplication.

Embodiment 522

The pharmaceutical composition of embodiment 521, comprising a peptide.

Embodiment 523

The pharmaceutical composition of embodiment 522, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 524

The pharmaceutical composition of embodiment 523, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 525

The pharmaceutical composition of embodiment 523, wherein the peptidecomprises P13.

Embodiment 526

The pharmaceutical composition of embodiment 523, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 527

The pharmaceutical composition of embodiment 526, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 528

The pharmaceutical composition of embodiment 521, wherein the topicalapplication comprises application to the skin, hair, outer ear, tympanicmembrane, buccal cavity, or sublingual cavity.

Embodiment 529

The pharmaceutical composition of embodiment 528, comprising a peptide.

Embodiment 530

The pharmaceutical composition of embodiment 529, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 531

The pharmaceutical composition of embodiment 530, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 532

The pharmaceutical composition of embodiment 530, wherein the peptidecomprises P13.

Embodiment 533

The pharmaceutical composition of embodiment 530, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 534

The pharmaceutical composition of embodiment 533, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 535

The pharmaceutical composition of embodiment 528, wherein the topicalapplication comprises application to the tympanic membrane.

Embodiment 536

The pharmaceutical composition of embodiment 535, comprising a peptide.

Embodiment 537

The pharmaceutical composition of embodiment 536, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 538

The pharmaceutical composition of embodiment 537, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 539

The pharmaceutical composition of embodiment 537, wherein the peptidecomprises P13.

Embodiment 540

The pharmaceutical composition of embodiment 537, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 541

The pharmaceutical composition of embodiment 540, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 542

The pharmaceutical composition of embodiment 535, wherein theapplication to the tympanic membrane comprises the application of eardrops to the tympanic membrane.

Embodiment 543

The pharmaceutical composition of embodiment 542, comprising a peptide.

Embodiment 544

The pharmaceutical composition of embodiment 543, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 545

The pharmaceutical composition of embodiment 544, wherein the peptidecomprises P13, a peptide comprising P13 and a poly-argenine domain, or apeptide of Table 1.

Embodiment 546

The pharmaceutical composition of embodiment 544, wherein the peptidecomprises P13.

Embodiment 547

The pharmaceutical composition of embodiment 544, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 548

The pharmaceutical composition of embodiment 547, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 549

A method of treating ear inflammation in an animal in need or wantthereof, the method comprising administering to the animal thepharmaceutical composition of embodiment 507.

Embodiment 550

The method of embodiment 549, wherein the ear inflammation comprisesotitis media.

Embodiment 551

The method of embodiment 550, further comprising wherein thepharmaceutical composition is applied via topical application.

Embodiment 552

The method of embodiment 551, wherein the topical application comprisesapplication to the skin, hair, outer ear, tympanic membrane, buccalcavity, or sublingual cavity.

Embodiment 553

The method of embodiment 552, wherein the topical application comprisesapplication to the tympanic membrane.

Embodiment 554

The method of embodiment 553, wherein the application to the tympanicmembrane comprises the application of ear drops to the tympanicmembrane.

Embodiment 555

The method of embodiment 549, 550, 551, 552, 553, or 554, wherein thepharmaceutical composition comprises P13, a P13 variant, derivative,stereoisomer, or analogue, or a peptide of Table 1.

Embodiment 556

The method of embodiment 555, wherein the P13 variant, derivative,stereoisomer, or analogue comprises P13 and a poly-argenine domain.

Embodiment 557

The method of embodiment 555, wherein the pharmaceutical compositioncomprises P13.

Embodiment 558

The pharmaceutical composition of embodiment 555, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 559

The method of embodiment 558, wherein the peptide comprises astereoisomer of P13 wherein all the amino acid residues of thestereoisomer comprise the D-configuration.

Embodiment 560

A method of treating ear inflammation in an animal, the methodcomprising administering a peptide to the animal, wherein the peptidecomprises P13, a P13 variant, derivative, stereoisomer, or analogue, ora peptide of Table 1.

Embodiment 561

The method of embodiment 560, wherein the ear inflammation comprisesotitis media.

Embodiment 562

The method of embodiment 561, wherein the administering of the peptidecomprises application to the skin, hair, outer ear, tympanic membrane,buccal cavity, or sublingual cavity.

Embodiment 563

The method of embodiment 562, wherein the topical application comprisesapplication to the tympanic membrane, optionally via ear drops.

Embodiment 564

The method of embodiment 563, wherein the peptide comprises P13.

Embodiment 565

The method of embodiment 564, wherein the animal is human.

Embodiment 566

The method of embodiment 563, wherein the peptide comprises P13 and apoly-argenine domain.

Embodiment 567

The method of embodiment 566, wherein the animal is human.

Embodiment 568

The method of embodiment 563, wherein the peptide comprisesDIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 378).

Embodiment 569

The method of embodiment 568, wherein the animal is human.

Embodiment 570

The method of embodiment 563, wherein the peptide comprises astereoisomer of P13, wherein one or more amino acid residues of thestereoisomer comprise the D-configuration.

Embodiment 571

The method of embodiment 570, wherein the peptide comprises astereoisomer of P13 wherein all the amino acid residues of thestereoisomer comprise the D-configuration.

Embodiment 572

The method of embodiment 571, wherein the animal is human.

Embodiment 573

The method of embodiment 563, wherein the peptide comprises an analogueof P13, wherein one or more amino acid residues of the analogue comprisethe D-configuration.

Embodiment 574

The method of embodiment 573, wherein the peptide comprises an analogueof P13, wherein all the amino acid residues of the analoguecorresponding to a residue of P13 comprise the D-configuration, and apoly-argenine domain.

Embodiment 575

The method of embodiment 574, wherein the animal is human.

Embodiment 576

The method of embodiment 563, wherein the peptide comprises:

(SEQ ID NO: 183) D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg.

Embodiment 577

The method of embodiment 576, wherein the animal is human.

Embodiment 578

The method of embodiment 573, wherein the peptide comprises an analogueof P13, wherein all the amino acid residues of the analoguecorresponding to a residue of P13 comprise the D-configuration, and apoly-argenine domain comprising at least one amino acid residue of theD-configuration.

Embodiment 579

The method of embodiment 578, wherein the animal is human.

Embodiment 580

The method of embodiment 563, wherein the peptide comprises:

(SEQ ID NO: 184) D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg.

Embodiment 581

The method of embodiment 580, wherein the animal is human.

Embodiment 582

A peptide comprising the amino acid sequence:D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg(SEQ ID NO: 183).

Embodiment 583

A peptide comprising the amino acid sequence:D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg(SEQ ID NO: 184).

Embodiment 584

A peptide comprising the amino acid sequence:D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile (SEQID NO: 185).

Embodiment 585

A peptide comprising the amino acid sequence:Asp-Ile-Val-Lys-Leu-Thr-Val-Tyr-Asp-Cys-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg(SEQ ID NO: 186).

Embodiment 586

A pharmaceutical composition comprising a peptide, the peptidecomprising:

P13 or a variant, derivative, stereoisomer, or analogue thereof; or

a peptide of Table 1,

wherein the composition is used for the treatment of inflammation of theskin, joints, muscular tissue, or connective tissue in a subject.

Embodiment 587

The pharmaceutical composition of embodiment 586, wherein theinflammation comprises arthritis, dermatitis, Lupus erythematosus, orpsoriasis.

Embodiment 588

The pharmaceutical composition of embodiment 587, wherein the arthritiscomprises osteoarthritis, rheumatoid arthritis, septic arthritis, gout,pseudo-gout, juvenile idiopathic arthritis, Still's disease, orankylosing spondylitis.

Embodiment 589

The pharmaceutical composition of embodiment 587, wherein the dermatitiscomprises spongiotic dermatitis, childhood eczema, allergic contactdermatitis, seborrhoeic dermatitis, dyshidrotic dermatitis, urticaria,vesicular or bullous dermatitis, or papular urticaria.

Embodiment 590

The pharmaceutical composition of embodiment 587, wherein the psoriasiscomprises plaque psoriasis, flexural psoriasis, guttate psoriasis,pustular psoriasis, nail psoriasis, psoriatic arthritis, orerythrodermic psoriasis.

Embodiment 591

The pharmaceutical composition of embodiment 586, further comprising oneor more pharmaceutically-acceptable excipients.

Embodiment 592

The pharmaceutical composition of embodiment 586, wherein the peptidecomprises P13 or a variant, derivative, stereoisomer, or analoguethereof.

Embodiment 593

The pharmaceutical composition of embodiment 592, wherein the peptidecomprises P13, or a peptide comprising P13 and a poly-argenine domain.

Embodiment 594

The pharmaceutical composition of embodiment 592, wherein the peptidecomprises P13.

Embodiment 595

The pharmaceutical composition of embodiment 592, wherein the peptidecomprises a stereoisomer of P13, wherein one or more amino acid residuesof the stereoisomer comprise the D-configuration.

Embodiment 596

The pharmaceutical composition of embodiment 595, wherein the peptidecomprises a stereoisomer of P13 wherein all the amino acid residues ofthe stereoisomer comprise the D-configuration.

Embodiment 597

The pharmaceutical composition of embodiment 592, wherein the peptidecomprises P13 and a poly-argenine domain.

Embodiment 598

The pharmaceutical composition of embodiment 597, wherein the peptidecomprises DIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 378).

Embodiment 599

The pharmaceutical composition of embodiment 592, wherein the peptidecomprises an analogue of P13, wherein one or more amino acid residues ofthe analogue comprise the D-configuration.

Embodiment 600

The pharmaceutical composition of embodiment 599, wherein the peptidecomprises an analogue of P13, wherein all the amino acid residues of theanalogue corresponding to a residue of P13 comprise the D-configuration,and a poly-argenine domain.

Embodiment 601

The pharmaceutical composition of embodiment 599, wherein the peptidecomprises:D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg(SEQ ID NO: 183).

Embodiment 602

The pharmaceutical composition of embodiment 599, wherein the peptidecomprises:D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg(SEQ ID NO: 184).

Embodiment 603

A method of treating inflammation in an animal, the method comprisingadministering a peptide to the animal, wherein the peptide comprisesP13, a P13 variant, derivative, stereoisomer, or analogue, or a peptideof Table 1.

Embodiment 604

The method of embodiment 603, wherein the inflammation comprisesarthritis, dermatitis, Lupus erythematosus, meningitis, or psoriasis.

Embodiment 605

The method of embodiment 604, wherein the arthritis comprisesosteoarthritis, rheumatoid arthritis, septic arthritis, gout,pseudo-gout, juvenile idiopathic arthritis, Still's disease, orankylosing spondylitis.

Embodiment 606

The method of embodiment 604, wherein the dermatitis comprisesspongiotic dermatitis, childhood eczema, allergic contact dermatitis,seborrhoeic dermatitis, dyshidrotic dermatitis, urticaria, vesicular orbullous dermatitis, or papular urticaria.

Embodiment 607

The method of embodiment 604, wherein the psoriasis comprises plaquepsoriasis, flexural psoriasis, guttate psoriasis, pustular psoriasis,nail psoriasis, psoriatic arthritis, or erythrodermic psoriasis.

Embodiment 608

The method of embodiment 603, wherein the peptide comprises P13 or avariant, derivative, stereoisomer, or analogue thereof.

Embodiment 609

The method of embodiment 608, wherein the peptide comprises P13, or apeptide comprising P13 and a poly-argenine domain.

Embodiment 610

The method of embodiment 608, wherein the peptide comprises P13.

Embodiment 611

The method of embodiment 608, wherein the peptide comprises astereoisomer of P13, wherein one or more amino acid residues of thestereoisomer comprise the D-configuration.

Embodiment 612

The method of embodiment 611, wherein the peptide comprises astereoisomer of P13 wherein all the amino acid residues of thestereoisomer comprise the D-configuration.

Embodiment 613

The method of embodiment 608, wherein the peptide comprises P13 and apoly-argenine domain.

Embodiment 614

The method of embodiment 613, wherein the peptide comprisesDIVKLTVYDCIRRRRRRRRR (SEQ ID NO: 378).

Embodiment 615

The method of embodiment 608, wherein the peptide comprises an analogueof P13, wherein one or more amino acid residues of the analogue comprisethe D-configuration.

Embodiment 616

The method of embodiment 615, wherein the peptide comprises an analogueof P13, wherein all the amino acid residues of the analoguecorresponding to a residue of P13 comprise the D-configuration, and apoly-argenine domain.

Embodiment 617

The method of embodiment 615, wherein the peptide comprises:

(SEQ ID NO: 183) D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg.

Embodiment 618

The method of embodiment 615, wherein the peptide comprises:

(SEQ ID NO: 184) D-Asp-D-Ile-D-Val-D-Lys-D-Leu-D-Thr-D-Val-D-Tyr-D-Asp-D-Cys-D-Ile-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg.

Embodiment 619

The method of any of embodiments 603-618, wherein the animal is a human.

Embodiment 6120

The method of any of embodiments 603-618, wherein the administeringcomprises topical application.

REFERENCES

The following references, and those cited in the disclosure herein, arehereby incorporated herein in their entirety by reference.

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What is claimed is:
 1. A pharmaceutical peptide composition comprising:a peptide-transducing sequence conjugate, wherein the peptide comprisesSEQ ID NO: 337, and wherein the peptide is conjugated to the transducingsequence at the peptide C-terminus; and a pharmaceutically-acceptableexcipient, wherein the pharmaceutical composition is in a unit dosageform.
 2. The pharmaceutical peptide composition of claim 1, wherein thetransducing sequence is a poly-arginine sequence.
 3. The pharmaceuticalpeptide composition of claim 1, wherein the transducing sequencecomprises nine consecutive arginine residues (SEQ ID NO: 379).
 4. Thepharmaceutical peptide composition of claim 1, wherein the transducingsequence consists of nine consecutive arginine residues (SEQ ID NO:379).
 5. The pharmaceutical peptide composition of claim 1, wherein atleast one amino acid residue of the transducing sequence is a D-aminoacid residue.
 6. The pharmaceutical peptide composition of claim 1,wherein all the amino acid residues of the transducing sequence areD-amino acid residues.
 7. The pharmaceutical peptide composition ofclaim 1, wherein the peptide is SEQ ID NO:
 337. 8. The pharmaceuticalpeptide composition of claim 1, wherein the peptide-transducing sequenceconjugate is SEQ ID NO:
 151. 9. The pharmaceutical peptide compositionof claim 1, wherein the pharmaceutical peptide composition is in theform of an ointment, cream, liquid, gel, or salve.
 10. Thepharmaceutical peptide composition of claim 1, wherein thepharmaceutical peptide composition is in the form of a drop.
 11. Thepharmaceutical peptide composition of claim 1, wherein thepharmaceutical peptide composition is in the form of an aerosol, vapor,spray, or mist.
 12. A synthetic peptide of SEQ ID NO: 337, conjugated toa transducing sequence at the C-terminus; and wherein at least one aminoacid residue of the synthetic peptide is in the D-configuration.
 13. Thesynthetic peptide of claim 12, wherein the synthetic peptide is of SEQID NO:
 151. 14. A synthetic peptide-transducing sequence conjugate,wherein the synthetic peptide comprises SEQ ID NO: 337, and wherein thesynthetic peptide is conjugated to the transducing sequence at thepeptide C-terminus.
 15. The synthetic peptide-transducing sequenceconjugate of claim 14, wherein the synthetic peptide is SEQ ID NO: 337.16. The synthetic peptide-transducing sequence conjugate of claim 14,wherein the synthetic peptide-transducing sequence conjugate is SEQ IDNO:
 151. 17. The synthetic peptide-transducing sequence conjugate ofclaim 14, wherein the transducing sequence is a poly-arginine sequence.18. The synthetic peptide-transducing sequence conjugate of claim 14,wherein the transducing sequence comprises nine consecutive arginineresidues (SEQ ID NO: 379).
 19. The synthetic peptide-transducingsequence conjugate of claim 14, wherein the transducing sequenceconsists of nine consecutive arginine residues (SEQ ID NO: 379).
 20. Thesynthetic peptide-transducing sequence conjugate of claim 14, wherein atleast one amino acid residue of the transducing sequence is a D-aminoacid residue.
 21. The synthetic peptide-transducing sequence conjugateof claim 14, wherein all the amino acid residues of the transducingsequence are D-amino acid residues.